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Journal of Hazardous Materials 192 (2011) 1819– 1826

Contents lists available at ScienceDirect

Journal of Hazardous Materials

jou rn al h om epage: www.elsev ier .com/ loc ate / jhazmat

elective removal of 17�-estradiol with molecularly imprintedarticle-embedded cryogel systems

˙lker Koc , Gözde Baydemir, Engin Bayram, Handan Yavuz, Adil Denizli ∗

hemistry Department, Biochemistry Division, Hacettepe University, Ankara, Turkey

r t i c l e i n f o

rticle history:eceived 7 February 2011eceived in revised form 1 July 2011

a b s t r a c t

The selective removal of 17�-estradiol (E2) was investigated by using molecularly E2 imprinted (MIP) par-ticle embedded poly(hydroxyethyl methacrylate) (PHEMA) cryogel. PHEMA/MIP composite cryogel wascharacterized by FTIR, SEM, swelling studies, and surface area measurements. E2 adsorption studies were

ccepted 4 July 2011vailable online 12 July 2011

eywords:ryogelsolecular imprinting (MIP)

performed by using aqueous solutions which contain various amounts of E2. The specificity of PHEMA/MIPcryogel to recognition of E2 was performed by using cholesterol and stigmasterol. PHEMA/MIP cryogelexhibited a high binding capacity (5.32 mg/g polymer) and high selectivity for E2 in the presence ofcompetitive molecules, cholesterol (kE2/cholesterol = 7.6) and stigmasterol (kE2/Stigmasterol = 85.8). There is nosignificant decrease in adsorption capacity after several adsorption–desorption cycles.

7�-Estradiol

. Introduction

Environmental pollutants have many adverse effects on humannd wildlife [1]. Some sorts of these pollutants called endocrine dis-upters (EDs) cause reproductive system deformities on humans,ome developmental defects of children [2] and greatly elevatedisk of cancer [3]. Estradiol (E2 or 17�-estradiol), is the predomi-ant sex hormone present in females, is one of the most potent EDshich is commonly found in wastewaters and rivers [4].

Recent studies showed that conventional water treatmentethods are inefficient to remove EDs from surface and drinkingater due to their trace concentrations [5,6]. Conventional meth-

ds using advanced oxidation processes [7], ozonation [8], sandltration [9], chlorination [10], nanofiltration and reverse osmosisystems [11,12] seem suitable for removing the EDs, but they areot specific, require high energy demands and expensive. In recentears, a novel method is improved to remove EDs from water bysing molecularly imprinted polymers (MIP). This method is notnly suitable for low EDs concentration levels but also suitable forelective removal of E2 from mixed interfering substances [13].IPs are alternative sorbents to the conventional methods with

heir high selectivity, cost efficiency, reusability, easy preparationnd uses [14].

Molecular imprinting technique involves polymerization ofunctional monomers and a cross-linker around a template. Theemoval of the template leaves specific binding sites in the poly-

∗ Corresponding author. Tel.: +90 312 2977983; fax: +90 312 2992163.E-mail address: denizli@hacettepe.edu.tr (A. Denizli).

304-3894/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.jhazmat.2011.07.017

© 2011 Elsevier B.V. All rights reserved.

mer with the shape and the orientation of the functional groupscomplementary to those of the template molecule. The interactionsbetween the template and recognition sites of the polymer can benon-covalent such as hydrogen bonds, hydrophobic/electrostaticinteractions, or reversible covalent interactions [14–20].

Cryogels are novel polymeric structures with many advan-tages including large pores, short diffusion path, low pressuredrop and very short residence time [21–23]. But, due to the exis-tence of large pores within the cryogel, the adsorption capacityfor the biomolecules is low [24]. In actual bioseparation pro-cesses, it is of great importance to improve the binding capacity ofsupermacroporous cryogel. Therefore, particle embedding wouldbe a useful improvement mode to use in the preparation ofnovel composite cryogels for increasing surface area [25–27].This approach makes use of a combinational selection strategy toenhance adsorption capacity [28]. We report herein the selective E2removal with poly(hydroxyethyl methacrylate) (PHEMA) cryogelwith embedded E2-imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-tyrosine methylester) particles [PHEMA/MIPcomposite cryogel]. E2 adsorption and selectivity studies versusother competitive substances such as cholesterol and stigmasterolare reported here. Finally, repeated use of the PHEMA/MIP compos-ite cryogel has been also studied.

2. Experimental

2.1. Materials

E2, cholesterol, stigmasterol, l-tyrosine methylester andmethacryloyl chloride were purchased from Sigma (St. Louis,

1 ous Ma

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2

iMtm1dttlcre

2p

M(crwwEeiPtrmgtspu

2

imwami(i

820 I. Koc et al. / Journal of Hazard

SA). Hydroxyethyl methacrylate (HEMA) and ethylene glycolimethacrylate (EGDMA) were obtained from Sigma (St. Louis,SA), distilled under reduced pressure in the presence of hydro-uinone inhibitor and stored at 4 ◦C until use. Ammoniumersulfate (APS), N,N,N′,N′-tetramethylene diamine (TEMED) andPLC grade methanol were also obtained from Sigma. All otherhemicals were of reagent grade and purchased from Merck AGDarmstadt, Germany). All water used in the experiments wasurified using a Barnstead (Dubuque, IA) ROpure LP® reverse osmo-is unit with a high flow cellulose acetate membrane (Barnstead2731) followed by a Barnstead D3804 NANOpure® organic/colloid

emoval and ion exchange packed-bed system. Buffer and sampleolutions were prefiltered through a 0.2 �m membrane (Sartorius,ottingen, Germany). All glassware was extensively washed withilute nitric acid before use.

.2. Synthesis of N-methacryloyl-(l)-tyrosine methylester (MAT)

The MAT was selected as the functional monomer for E2mprinting. Details of the preparation and characterization of the

AT were reported elsewhere [29]. The following experimen-al procedure was applied for the synthesis of MAT. l-Tyrosine

ethylester (5 g) and hydroquinone (0.2 g) were dissolved in00 mL of dichloromethane solution. This solution was cooledown to 0 ◦C. Triethylamine (12.7 g) was added to the solu-ion and 5.0 mL of methacryloyl chloride was poured slowly intohis solution while stirring at room temperature for 2 h, fol-owed by extraction of hydroquinone and unreacted methacryloylhloride with ethyl acetate. Liquid phase was evaporated in aotary evaporator. The residue (i.e., MAT) was crystallized in anther–cyclohexane mixture and then dissolved in ethyl alcohol.

.3. Preparation of E2-imprinted poly(HEMA-MAT) [MIP]articles

In the first part, MAT–E2 complex was prepared. Briefly,AT (1 mmol) was dissolved in 2 mL of acetonitrile, then E2

1 mmol) was added in this solution. Toluene and ethylene gly-ol dimethacrylate (EGDMA) were included in the polymerizationecipe as the pore former and cross-linker, respectively. TEMEDas used as the activator. APS (20 mg) and TEMED (100 �L)ere dissolved in the mixture of monomers (HEMA: 1.0 mmol,

2–MAT complex: 500 �L, EGDMA: 20 mmol) and porogenic dilu-nt (toluene: 500 �L). The polymerization mixture was pouredn a glass tube and sealed after purging with nitrogen for 2 min.olymerization was completed in 10 min at room temperature. Athe end of the polymerization reaction, soluble components wereemoved from the polymer by repeated decantation with water andethanol. E2-imprinted monolith was smashed and the particles

rounded. The grounded polymers were sieved and the fractionhat fall in the size range of 5–10 �m were used throughout thetudy. Non-imprinted particles (NIP) were prepared, as contrololymer, in the same manner as that previously described, withoutsing E2 as template.

.4. Production of PHEMA cryogel with MIP/NIP particles

Production of the PHEMA cryogel with embedded MIP particless described below. Briefly, monomers (1.6 mL HEMA and 0.3 g N,N-

ethylene-bis(acrylamide) (MBAAm) were dissolved in deionizedater (5 mL) and the mixture was degassed under vacuum for

bout 5 min to eliminate soluble oxygen. Total concentration of

onomers was 12% (w/v). The cryogel was produced by free rad-

cal polymerization initiated by TEMED and APS. After adding APS25 �L, 1% (w/v) of the total monomers) the solution was cooledn an ice bath for 2–3 min. Then, TEMED (20 mg, 1% (w/v) of the

terials 192 (2011) 1819– 1826

total monomers) was added and the reaction mixture was stirredfor 1 min. In this step, the MIP particles (150 mg) were mixed withthe polymerization mixture. Then, the reaction mixture was pouredbetween two glass plates separated with 1.5 mm thick spacers. Thepolymerization solution in the plates was frozen at −16 ◦C for 24 hand then thawed at room temperature. The resulting cryogel sheetswere cut into circular pieces (2 cm diameter) with a perforator. Allof the experiments were performed with a 2 cm diameter columnincluding three pieces of the cryogel membrane sheet. After wash-ing with 200 mL of water, elution solution (acetonitrile: methanol(70:30 (v/v)) was passed through the PHEMA/MIP composite cryo-gel at room temperature for 3 h. This procedure was repeated untilno E2 leakage was observed from the polymeric structure to thewash solution. The E2 free composite cryogel was washed withethanol and water at room temperature for 12 h and the cryogelswere stored in buffer containing 0.02% sodium azide at 4 ◦C untiluse.

2.5. Characterization of cryogel

Water uptake ratio (S) of the PHEMA/MIP composite cryogel wasdetermined in distilled water. The experiment was conducted asfollows: initially dry cryogel was carefully weighed before beingplaced in a 40 mL vial containing distilled water. The vial was putinto an isothermal water bath with a fixed temperature (25 ◦C) for2 h. The sample was taken out from the water, wiped using a filterpaper, and weighed. In different time intervals the weight of thecryogel was recorded. After 24 h the final weight of cryogel wasrecorded. The water uptake ratio of the cryogel was calculated as

S =[

(Ws − W0)W0

]× 100 (1)

where W0 and Ws are the weights of cryogel before and after uptakeof water, respectively. The gelation yield was determined as fol-lows: the swollen cryogel sample (1 mL) was put in an oven at 60 ◦Cfor drying. After drying till constant weight, the mass of the driedsample was determined (mdried). The gel fraction yield was definedas

Gelation yield =(

mdried

mt

)× 100% (2)

where mt is the total mass of the monomers in the feed mixture.The total volume of macropores in the swollen cryogel was roughlyestimated by weighing the sample (msqueezed gel) after squeezingthe free water from the swollen gel matrix, and then the porositywas calculated as

Porosity (%) = (mswollen gel − msqueezed gel)mswollen gel

× 100% (3)

All measurements were done in triplicate and the average valuesare presented.

The flow-rate of water passing through the column was mea-sured at the constant hydrostatic pressure equal to 100 cm watercolumn corresponding to a pressure of ca. 0.01 MPa. At least threemeasurements were done for each sample.

Porosity of the polymer sample was measured by the nitro-gen sorption technique, performed on Flowsorb II, (MicromeriticsInstrument Corporation, Norcross, USA). The specific surface areaof composite cryogel in dry state was determined by multipointBrunauer– Emmett–Teller (BET) apparatus (Quantachrome, Nova2200E, USA). 0.5 g of sample was placed in a sample holder and

degassed in a N2-gas stream at 15 ◦C for 1 h. Adsorption of the gaswas performed at 21 ◦C and desorption was performed at roomtemperature. Values obtained from desorption step was used forthe specific surface area calculation.

us Ma

tfioppltwsi4gn

MsmdMt

s(

2

btWdTptusiiifa

w

L

L

wt

2

msMTrtmoccu

I. Koc et al. / Journal of Hazardo

The morphology of a cross section of the cryogel was inves-igated by scanning electron microscope (SEM). The sample wasxed in 2.5% glutaraldehyde in 0.15 M sodium cacodylate buffervernight, post-fixed in 1% osmium tetroxide for 1 h. Then the sam-le was dehydrated stepwise in ethanol and transferred to a criticaloint drier temperated to 10 ◦C where the ethanol was changed for

iquid carbon dioxide as transitional fluid. The temperature washen raised to 40 ◦C and the pressure to ca. 100 bar. Liquid CO2as transformed directly to gas uniformly throughout the whole

ample without heat of vaporization or surface tension forces caus-ng damage. Release of the pressure at a constant temperature of◦C resulted in dried cryogel sample. Finally, it was coated withold–palladium (40:60) and examined using a JEOL JSM 5600 scan-ing electron microscope (JEOL, JSM 5600, Tokyo, Japan).

Fourier transform infrared (FTIR) spectra of MAT, E2 and theAT–E2 complex, and MIP particles were obtained using a FTIR

pectrophotometer (FTIR 8000 Series, Shimadzu, Japan). E2, MATonomer, MAT–E2 complex, were dried in a vacuum oven. The

ry sample (0.1 g) was thoroughly mixed with KBr (0.1 g, IR Grade,erck, Germany), and pressed into a pellet form and the FTIR spec-

rum was then recorded.To evaluate the amount of MAT into the cryogel structure, it was

ubjected to elemental analysis using a Leco Elemental AnalyzerModel CHNS-932, USA).

.6. Assay of steroids

The detection of E2, cholesterol and stigmasterol was followedy using high performance liquid chromatography (HPLC) sys-em (Ultimate-3000, Dionex, USA) equipped with LPG-3000 pump,

PS-3000 autosampler, TCC-3000 column department, PDA-3000etector and column (Kromasil 100-5, Length/I.D; 150/4.6 mm).he acetonitrile–methanol–water mixture was used as mobilehase. Mobile phases A, B and C were water, methanol and acetoni-rile respectively. The chromatographic separation was performedsing a linear gradient at 0.5 mL/min flow rate. A linear gradienttarted from 10% B, 80% C and 10% A in 5 min, continued withncreasing B from 10% to 65% and decreasing C from 80% to 35%n 1 min and finished in 20 min. 100 �L of steroid solution wasnjected into the column. The absorbance was monitored at 280 nmor E2, cholesterol and stigmasterol. The separation was performedt ambient temperature.

The limit of detection (LOD) and limit of quantification (LOQ)ere calculated from the following equations:

OD = 3.3 × s

m(4)

OQ = 10 × s

m(5)

here s is the standard deviation of response and m is the slope ofhe corresponding calibration curve.

.7. E2 removal studies from aqueous media

The E2 adsorption studies were carried out in surface waterimicking medium with a continuous system, in a recirculation

ystem equipped with a water jacket for temperature control. TheIP composite cryogel was washed with 30 mL of water for 30 min.

hen, the E2 solution was pumped through the column underecirculation for 2 h. The adsorption was followed by using spec-rophotometer (UV-1601, Shimadzu, Japan) the absorbance was

onitored at 280 nm for E2. Effects of E2 concentration, flow-rate

n the adsorption amount were studied. The effect of the initial con-entration of E2 on adsorption capacity was studied by changing theoncentration of E2 between 50 and 100 �g/L by using large vol-me (2 mL) injection with optional loop and 5–50 mg/L. The effect

terials 192 (2011) 1819– 1826 1821

of flow rate on adsorption capacity was investigated at differentflow rates in the range of 0.5–4.0 mL/min when pumped throughthe cryogel column under recirculation for 2.0 h with 30 mg/L of E2solution in methanol–water (10:90 (v/v)). The amount of E2 adsorp-tion per unit mass of dry cryogel was calculated using the massbalance. Each data collected is average of three determinations.

2.8. Selectivity experiments

In order to show the specificity of PHEMA/MIP composite cryo-gel, competitive adsorption of cholesterol (MW: 386 g/mol) andstigmasterol (MW: 412.7 g/mol) was also studied. E2 aqueoussolution was overloaded with cholesterol and stigmasterol andapplied on PHEMA/MIP column. E2, cholesterol and stigmasterolwere added in methanol:water (10:90 (v/v)) solution and soni-cated for 10 min at room temperature. After attaining adsorptionequilibrium, the concentrations of cholesterol and stigmasterol inthe remaining solution were measured by the same HPLC systemexplained in Section 2.6.

The distribution coefficient (Kd) for cholesterol and stigmasterolwith respect to E2 was calculated by

Kd =[

(Ci − Cf)Cf

]× V

m(6)

where Kd represents the distribution coefficient (mL/g); Ci and Cfare initial and final concentrations of E2 (mg/mL), respectively. Vis the volume of solution (mL) and m is the weight of the cryogelcolumn (g). The selectivity coefficient (k) for the binding of E2 inthe presence of other competitive molecules can be obtained frombinding data according to

k = Kd

Kd(7)

where k is the selectivity coefficient and X represents cholesterolor stigmasterol. A comparison of the k values of the PHEMA/MIPcryogel with those cholesterol or stigmasterol allows an estima-tion of the effect of imprinting on selectivity. A relative selectivitycoefficient k′ can be defined as Eq. (8);

k′ = kimprinted

kcontrol(8)

2.9. Elution and repeated use

In order to show the stability and reusability of the PHEMA/MIPcomposite cryogel, the adsorption–elution cycle was repeated10 times using the same PHEMA/MIP composite cryogel ina continuous experimental set-up. For sterilization after oneadsorption–elution cycle, the cryogel was washed with 50 mMNaOH solution for 30 min. After this procedure, cryogel was washedwith distilled water for 30 min. Elution of E2 was studied with ace-tonitrile: methanol (70:30 (v/v)) solution. PHEMA/MIP compositecryogel was contacted with elution medium for 2 h at room tem-perature. The final E2 concentration in the elution medium wasmeasured as described above. The elution ratio was calculated fromthe amount of E2 adsorbed on the cryogel and the final E2 concen-tration in the elution medium.

3. Results and discussion

3.1. Characterization of composite cryogels

Table 1 presents the swelling properties and linear flow resis-tance (at hydrostatic pressure, ca. 0.01 mPa) of both PHEMA/MIPand PHEMA/NIP cryogels, respectively. Cryogels have superma-cropores, as a consequence of this; the flow rate through the gel

1822 I. Koc et al. / Journal of Hazardous Materials 192 (2011) 1819– 1826

Table 1Swelling properties and flow resistance of cryogels.

Polymer Monomer concentration (%) Gel yield (%) Swelling ratio (%) Swelling degree (g H2O/g polymer) Macroporosity (%) Flow rate (cm h−1)

PHEMA/MIP 10 90.2 87 8.1 68 554

m5Na8e(

PHEMA/NIP 10 91 85PHEMA 10 94.2 90.4

atrix is high. PHEMA/MIP showed the maximum flow rate at54 cm h−1 and NIP showed a flow rate at 548 cm h−1. Both MIP,IP cryogels were produced with high gelation yield (about 90%)nd had similar swelling properties with a swelling degree of about

.0 g H2O/g polymer as compared to cryogels prepared by Baydemirt al. (about 6.1 g H2O/g polymer) [30] and Derazshamshir et al.about 10 g H2O/g polymer) [31].

Fig. 1. FTIR spectra of (A) E2, MAT monomer and MAT–E2 c

8.2 65 5489.2 78 765

FTIR spectra of the MAT monomer, E2, and MAT–E2 complex areshown in Fig. 1A. FTIR spectrum of pure E2 has characteristic peaks,which include H bonded O–H at 3438 and 3222 cm−1 and stronghydrocarbon peaks around 2900 cm−1. Stretching vibration bands

are at 1610 and 927 cm−1. FTIR spectrum of MAT has the char-acteristic stretching vibration of amide I and amide II absorptionbands at 1653 cm−1 and 1516 cm−1, a carbonyl band at 1733 cm−1,

omplex. (B) PHEMA/MIP polymer and MIP particles.

I. Koc et al. / Journal of Hazardous Materials 192 (2011) 1819– 1826 1823

Table 2Specific surface area of the polymers (m2/g).

Specific surface area (m2/g)

aMhttc3FaicFia

tFntwsafli

wat

duidM

3

Pf

PHEMA 25.0PHEMA/MIP 123.2PHEMA/NIP 115.3

nd an aromatic C–H band at 809 cm−1. The functional monomerAT is expected to interact with E2 through hydrogen bonds and

ydrophobic interactions through an aromatic ring. Furthermore,he aromatic peak at 801 cm−1 of MAT monomer shifts upfieldo 809 cm−1, because of hydrophobic interactions. The E2–MATomplex has also characteristic peaks of H bonded O–H bond at559 cm−1, amide I at 1654 cm−1 and amide II at 1515 cm−1.TheTIR spectrum of the MAT monomer and of the E2–MAT complexre almost same, without shifts in wave numbers of related peaks,t can be concluded that the characteristic peaks of MAT monomerover E2’s characteristic peaks in the E2–MAT complex spectrum.TIR spectrum of MIP particles and PHEMA/MIP polymer is shownn Fig. 1B. All include H bonded O–H at around 3430 and 3000 cm−1

nd strong hydrocarbon peaks around 2950 cm−1.The SEM images of the internal structures of the MIP par-

icles embedded PHEMA/MIP composite cryogel are shown inig. 2. PHEMA/MIP composite cryogel has continuous intercon-ected pores (10–100 �m in diameter) that provide high flow rateshrough the channels. SEM images showed that the MIP particlesere uniformly distributed into the PHEMA cryogel network. Pore

ize of the matrix is much larger than the size of the E2 molecules,llowing them to enter easily through the pores of the convectiveow of the water through the pores; the mass transfer resistance

s practically negligible.The specific surface area of the PHEMA/MIP composite cryogel

as determined to be 123.2 m2/g polymer and the specific surfacerea of the PHEMA/NIP composite cryogel column was determinedo be 115.3 m2/g (Table 2).

The incorporations of the MAT for MIP and NIP particles wereetermined to be 78 �mol/g and 72 �mol/g polymer, respectively,sing nitrogen stoichiometry. Note that HEMA and other polymer-

zation ingredients do not contain nitrogen. This nitrogen amount,etermined by elemental analysis, comes from only incorporatedAT groups into the polymeric structure.

.2. Removal of template

The acetonitrile–water solution was passed through theHEMA/MIP composite cryogel for the removal of the templaterom the cavities of MIP particles at room temperature for 3 h. Fig. 3

Fig. 2. SEM images of PHEMA/MIP composite cryogel.

Fig. 3. The chromatogram of the E2 released from the PHEMA/MIP composite cryogel.

1824 I. Koc et al. / Journal of Hazardous Materials 192 (2011) 1819– 1826

Table 3Statistical evaluation of the calibration data of E2 by HPLC.

Sample Linearity range(�g mL−1)

Slope Intercept S.D. of slope S.D. ofintercept

Correlationcoefficient

Detection limit(�g mL−1)

Quantitation limit(�g mL−1)

E2 3–20 3.124 0.030 0.069 0.853 0.999 0.768 2.562

F0

sPoda

dd0

3

3

tTc3icpahw

3

itrdtitiflEiei

ig. 4. Effect of equilibrium E2 concentration on adsorption amount: Flow-rate:.5 mL/min; T: 25 ◦C, Time: 2 h.

hows the decrease of amount of template E2 released from theHEMA/MIP polymer. The peaks labeled as 1–6 shows the amountf E2 released from the PHEMA/MIP cavities. After 6 elution proce-ure, no significant E2 peak was observed which means that almostll amount of E2 was removed from PHEMA/MIP composite cryogel.

The E2 concentration was detected by HPLC. The calibrationata was constructed by the plotting of absorbance versus stan-ard concentration (Table 3). The LOD for E2 was determined as.768 �g mL−1.

.3. E2 adsorption studies

.3.1. Effect of equilibrium concentration of E2Fig. 4 shows the equilibrium concentration of E2 dependence of

he adsorbed amount of E2 onto the PHEMA/MIP composite cryogel.he amount of adsorbed E2 increased with increasing E2 initial con-entration, and a saturation value is achieved at E2 concentration of0 mg/L which represents saturation of the accessible binding cav-

ties on the PHEMA/MIP composite cryogel. Maximum adsorptionapacity was 5.32 mg/g polymer. The MIP particle embedded com-osite cryogel play the significant role for binding E2 molecules, as

result of that PHEMA/MIP composite cryogel showed a 7 timesigher capacity to adsorb the E2 in water than the PHEMA cryogelith NIP particles (0.8 mg/g polymer) (Fig. 4).

.3.2. Effect of flow rateThe amounts of adsorbed E2 at different flow-rates are given

n Fig. 5. Results show that the E2 adsorption capacity ontohe PHEMA/MIP composite cryogel decreased when the flow-ate through the column increased. The amount of adsorbed E2ecreased significantly from 5.32 mg/g to 3.2 mg/g polymer withhe increase of the flow-rate from 0.5 mL/min to 4.0 mL/min. Thiss due to decrease in contact time between the E2 molecules andhe composite cryogel at higher flow-rates. These results are alson agreement with those referred to the literature [32]. When theow-rate decreases, the contact time in the column is longer. Thus,2 molecules have more time to recognize the E2 molecular cavities

n embedded MIP particles in the PHEMA cryogel structure and tonter to the molecular cavities; hence a higher adsorption amounts obtained.

Fig. 5. Effect of flow-rate onto the adsorption amount: E2 concentration: 30 mg/L;T: 25 ◦C.

3.4. Adsorption isotherm

Modeling of the equilibrium adsorption data has been doneusing the Langmuir and Freundlich isotherms [33]. A good fit isthe Langmuir isotherm (7),

Q = Qmax b Ceq

(1 + b Ceq)(9)

where q is the Langmuir monolayer adsorption capacity (mg/g),Ceq is the equilibrium E2 concentration (mg/mL), and b is the Lang-muir adsorption equilibrium constant, which indicates monolayeradsorption. The other well-known isotherm, which is frequentlyused to describe adsorption behavior, is the Freundlich isotherm(8):

q = KF C1/neq (10)

where KF is the Freundlich adsorption constant (mg/mL), Ceq isthe equilibrium E2 concentration (mg/mL), and n is the Freundlichexponent which represents the heterogeneity of the system. TheFreundlich isotherm describes reversible adsorption and is notrestricted to the formation of the monolayer. A more homogeneoussystem will have n value approaching unity while a more hetero-geneous system will have an n value approaching zero [34]. InTable 4, the experimental adsorption behavior was compared withLangmuir and Freundlich adsorption isotherms. The experimentaldata tend to be better fitted with Langmuir rather than Freundlichisotherm, since the correlation coefficient (R2) was high (0.98). Themaximum amount of adsorption (5.32 mg/g) obtained from exper-imental results is also close to the calculated Langmuir adsorptioncapacity (5.93 mg/g). The Langmuir and Freundlich adsorption con-stants with the correlation coefficients are given in Table 4. It canbe concluded that the adsorption of E2 onto PHEMAH/MIP cryogelis a monolayer adsorption.

The factors affecting adsorption mechanism, such as mass trans-fer and the binding itself were investigated using two differentkinetic models. The pseudo-first order and pseudo-second order

equations can be used in this case assuming that the measuredconcentrations are equal to adsorbent surface concentration. Thefirst-order rate expression of Lagergren [35] is one of the most

I. Koc et al. / Journal of Hazardous Materials 192 (2011) 1819– 1826 1825

Table 4Langmuir and Freundlich adsorption isotherm constants.

Experimental Langmuir constants Freundlich constants

qex (mg/L) qmax (mg/g) b (mL/mg) R2 KF n R2

5.32 5.93 0.00 0.98 36.23 2.3 0.75

Table 5The first and second order kinetic constants for PHEMAH/MIP.

Initial concentration (mg/L) Experimental First-order kinetic Second-order kinetic

qeq (mg/g) k1 (1/min) qeq (mg/g) R2 k2 (1/min) qeq (mg/g) R2

30 5.32 0.113 6.828 0.8413 0.00724 5.36 0.9498

res of

w(

l

w(rdkoapE

(

wttktatipiota

3

is

shows the adsorbed template and competitive molecules bothin PHEMA/MIP composite cryogel and PHEMA cryogel in mg/gpolymer.

Table 6Kd, k, and k′ values of cholesterol and stigmasterol with respect to E2.

Compound Polymer

NIP MIP

Fig. 6. Chemical structu

idely used for the adsorption of solute from a liquid solution, Eq.11):

og(qe − qt) = log(qe) − (k1t)2.303

(11)

here qe is the experimental amount of E2 adsorbed at equilibriummg/g); qt is the amount of E2 adsorbed at time t (mg/g); k1 is theate constant of the pseudo-first order adsorption (min−1). A linearependence of log(qe − qt) on t suggests the applicability of thisinetic model. Also, in many cases, the pseudo-first order equationf Lagergren does not fit well to the whole range of contact timend is generally applicable over the initial stage of the adsorptionrocesses. The pseudo-second order kinetic model is expressed byq. (12):

t

qt

)=

(1

k2q2eq

)+

(1

qeq

)t (12)

here k2 is the rate constant of the pseudo-second order adsorp-ion (g/mL min). If the pseudo-second order kinetics is applicable,he plot of t/q versus t should be linear. The pseudo-second orderinetic model is favorable, when the adsorption behavior overhe whole range of adsorption is in agreement with chemicaldsorption being the rate controlling step [36]. A comparison ofhe experimental adsorption capacity and the theoretical valuess presented in Table 5. The correlation coefficient for the linearlot of −log(qeq − qt) versus t for the pseudo-first order equation

s lower than the correlation coefficient for the pseudo-secondrder equation suggesting that the pseudo-second order adsorp-ion mechanism is predominant and that the overall rate of the E2dsorption process appeared to be controlled by binding kinetics.

.5. Selectivity studies

The specificity of PHEMA/MIP cryogel to recognition of E2 isnvestigated by using cholesterol and stigmasterol; molecules haveimilar chemical structure with E2. Fig. 6 shows the chemical struc-

competitor molecules.

tures of E2, cholesterol and stigmasterol. Molecular weight of E2is 272.4 g/mol while that of cholesterol is 386 g/mol and that ofstigmasterol is 412.7 g/mol.

Table 6 summarizes the Kd, k and k′ values in selectivitystudies. Distribution coefficient (Kd) value for the PHEMA/MIPcomposite cryogel is higher than that of the control PHEMA/NIPcryogel. Kd value showed an increase for E2 while it tendsto decrease for the competitor molecules, cholesterol and stig-masterol. The relative selectivity coefficient is an indicator toexpress binding affinity of recognition sites to the imprinted E2molecules [14,31]. The results show that relative selectivity coef-ficients of PHEMA/MIP composite cryogel for E2/cholesterol andE2/stigmasterol were 7.6 and 85.8 times greater than the PHEMA-NIP cryogel, respectively. Selectivity is dependent on the shape andsize memories of the imprinted cavities. The competitor moleculeswere less adsorbed by the PHEMA/MIP composite cryogel dueto linear chains in their chemical structures makes them morehydrophobic than E2. E2 has two hydroxyl groups so it consti-tutes strong hydrogen bonding through hydroxyl group on specificmonomer tyrosine in PHEMAT/MIP cavities. Nevertheless, the bind-ing specificity for E2 from PHEMA/MIP composite cryogel wassufficient for the recognition of E2 from other compounds. Fig. 7

Kd k Kd k k′

E2 89.2 851Cholesterol 41.6 2.1 52 16.34 7.6Stigmasterol 208.3 0.4 23 36.76 85.8

1826 I. Koc et al. / Journal of Hazardous Ma

Fig. 7. Adsorbed template and competitive molecules to the PHEMA/MIP andPHEMA/NIP composite cryogel: Flow rate: 1 mL/min; T: 25 ◦C.

Ft

3

c1aaNwciAa9

4

flccdppg

[

[

[

[

[[[[[

[[[

[

[

[

[

[

[

[

[

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ig. 8. Adsorption–elution cycle of PHEMA/MIP composite cryogel: E2 concentra-ion: 30 mg/L; flow rate: 0.5 mL/min; T: 25 ◦C.

.6. Elution and repeated use

In order to show the stability and reusability of the PHEMA/MIPomposite cryogel, the adsorption–elution cycle was repeated0 times using the same PHEMA/MIP composite cryogel in

continuous experimental set-up. For sterilization after onedsorption–elution cycle, the cryogel was washed with 50 mMaOH solution for 30 min. After this procedure, cryogel was washedith distilled water for 30 min. At the end of 10 adsorption–elution

ycle, there was no remarkable decrease in the E2 adsorption capac-ty (Fig. 8) and the percent removal ratio of E2 was found as 88%.s seen here that the PHEMA/MIP composite cryogel is very stable,nd maintain their adsorption capacity at almost constant value of6.3%.

. Conclusion

In this study PHEMAH/MIP composite cryogels combined highow path through interconnected supermacroporous structures ofryogels with high selectivity of the MIP particles PHEMAH/MIPomposite cryogels were produced and high selectivity for E2 was

emonstrated. The PHEMAH/MIP composite cryogels were pre-ared using 5–10 �m MIP particles. The incorporation of the MIParticles not only enhances the surface area of the PHEMA cryo-el (about 5 fold) but also increase the mechanical stability of

[

[[

terials 192 (2011) 1819– 1826

the monolithic cryogel column. The E2 removal from water withcompetitor molecules (E2 concentration 30 mg/L) was found as5.32 mg/g polymer with a considerably high removal ratio (88%).There is no significant decrease in adsorption capacity after severaladsorption–desorption cycles. The results presented here demon-strate that the PHEMAH/MIP composite cryogels can be used forthe recognition and selective removal of E2 molecules from waterwith a high E2 removal capacity.

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