Selective removal of 17β-estradiol with molecularly imprinted particle-embedded cryogel systems

Journal of Hazardous Materials 192 (2011) 1819–1826

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Selective removal of 17␤-estradiol with molecularly imprinted particle-embedded cryogel systems I˙ lker Koc¸, Gözde Baydemir, Engin Bayram, Handan Yavuz, Adil Denizli ∗ Chemistry Department, Biochemistry Division, Hacettepe University, Ankara, Turkey

a r t i c l e

i n f o

Article history: Received 7 February 2011 Received in revised form 1 July 2011 Accepted 4 July 2011 Available online 12 July 2011 Keywords: Cryogels Molecular imprinting (MIP) 17␤-Estradiol

a b s t r a c t The selective removal of 17␤-estradiol (E2) was investigated by using molecularly E2 imprinted (MIP) particle embedded poly(hydroxyethyl methacrylate) (PHEMA) cryogel. PHEMA/MIP composite cryogel was characterized by FTIR, SEM, swelling studies, and surface area measurements. E2 adsorption studies were performed by using aqueous solutions which contain various amounts of E2. The specificity of PHEMA/MIP cryogel to recognition of E2 was performed by using cholesterol and stigmasterol. PHEMA/MIP cryogel exhibited a high binding capacity (5.32 mg/g polymer) and high selectivity for E2 in the presence of competitive molecules, cholesterol (kE2/cholesterol = 7.6) and stigmasterol (kE2/Stigmasterol = 85.8). There is no significant decrease in adsorption capacity after several adsorption–desorption cycles. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Environmental pollutants have many adverse effects on human and wildlife [1]. Some sorts of these pollutants called endocrine disrupters (EDs) cause reproductive system deformities on humans, some developmental defects of children [2] and greatly elevated risk of cancer [3]. Estradiol (E2 or 17␤-estradiol), is the predominant sex hormone present in females, is one of the most potent EDs which is commonly found in wastewaters and rivers [4]. Recent studies showed that conventional water treatment methods are inefficient to remove EDs from surface and drinking water due to their trace concentrations [5,6]. Conventional methods using advanced oxidation processes [7], ozonation [8], sand filtration [9], chlorination [10], nanofiltration and reverse osmosis systems [11,12] seem suitable for removing the EDs, but they are not specific, require high energy demands and expensive. In recent years, a novel method is improved to remove EDs from water by using molecularly imprinted polymers (MIP). This method is not only suitable for low EDs concentration levels but also suitable for selective removal of E2 from mixed interfering substances [13]. MIPs are alternative sorbents to the conventional methods with their high selectivity, cost efficiency, reusability, easy preparation and uses [14]. Molecular imprinting technique involves polymerization of functional monomers and a cross-linker around a template. The removal of the template leaves specific binding sites in the poly-

mer with the shape and the orientation of the functional groups complementary to those of the template molecule. The interactions between the template and recognition sites of the polymer can be non-covalent such as hydrogen bonds, hydrophobic/electrostatic interactions, or reversible covalent interactions [14–20]. Cryogels are novel polymeric structures with many advantages including large pores, short diffusion path, low pressure drop and very short residence time [21–23]. But, due to the existence of large pores within the cryogel, the adsorption capacity for the biomolecules is low [24]. In actual bioseparation processes, it is of great importance to improve the binding capacity of supermacroporous cryogel. Therefore, particle embedding would be a useful improvement mode to use in the preparation of novel composite cryogels for increasing surface area [25–27]. This approach makes use of a combinational selection strategy to enhance adsorption capacity [28]. We report herein the selective E2 removal with poly(hydroxyethyl methacrylate) (PHEMA) cryogel with embedded E2-imprinted poly(hydroxyethyl methacrylateN-methacryloyl-(l)-tyrosine methylester) particles [PHEMA/MIP composite cryogel]. E2 adsorption and selectivity studies versus other competitive substances such as cholesterol and stigmasterol are reported here. Finally, repeated use of the PHEMA/MIP composite cryogel has been also studied. 2. Experimental 2.1. Materials

∗ Corresponding author. Tel.: +90 312 2977983; fax: +90 312 2992163. E-mail address: [email protected] (A. Denizli). 0304-3894/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2011.07.017

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

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USA). Hydroxyethyl methacrylate (HEMA) and ethylene glycol dimethacrylate (EGDMA) were obtained from Sigma (St. Louis, USA), distilled under reduced pressure in the presence of hydroquinone inhibitor and stored at 4 ◦ C until use. Ammonium persulfate (APS), N,N,N ,N -tetramethylene diamine (TEMED) and HPLC grade methanol were also obtained from Sigma. All other chemicals were of reagent grade and purchased from Merck AG (Darmstadt, Germany). All water used in the experiments was purified using a Barnstead (Dubuque, IA) ROpure LP® reverse osmosis unit with a high flow cellulose acetate membrane (Barnstead D2731) followed by a Barnstead D3804 NANOpure® organic/colloid removal and ion exchange packed-bed system. Buffer and sample solutions were prefiltered through a 0.2 ␮m membrane (Sartorius, Gottingen, Germany). All glassware was extensively washed with dilute nitric acid before use. 2.2. Synthesis of N-methacryloyl-(l)-tyrosine methylester (MAT) The MAT was selected as the functional monomer for E2 imprinting. Details of the preparation and characterization of the MAT were reported elsewhere [29]. The following experimental procedure was applied for the synthesis of MAT. l-Tyrosine methylester (5 g) and hydroquinone (0.2 g) were dissolved in 100 mL of dichloromethane solution. This solution was cooled down to 0 ◦ C. Triethylamine (12.7 g) was added to the solution and 5.0 mL of methacryloyl chloride was poured slowly into this solution while stirring at room temperature for 2 h, followed by extraction of hydroquinone and unreacted methacryloyl chloride with ethyl acetate. Liquid phase was evaporated in a rotary evaporator. The residue (i.e., MAT) was crystallized in an ether–cyclohexane mixture and then dissolved in ethyl alcohol. 2.3. Preparation of E2-imprinted poly(HEMA-MAT) [MIP] particles In the first part, MAT–E2 complex was prepared. Briefly, MAT (1 mmol) was dissolved in 2 mL of acetonitrile, then E2 (1 mmol) was added in this solution. Toluene and ethylene glycol dimethacrylate (EGDMA) were included in the polymerization recipe as the pore former and cross-linker, respectively. TEMED was used as the activator. APS (20 mg) and TEMED (100 ␮L) were dissolved in the mixture of monomers (HEMA: 1.0 mmol, E2–MAT complex: 500 ␮L, EGDMA: 20 mmol) and porogenic diluent (toluene: 500 ␮L). The polymerization mixture was poured in a glass tube and sealed after purging with nitrogen for 2 min. Polymerization was completed in 10 min at room temperature. At the end of the polymerization reaction, soluble components were removed from the polymer by repeated decantation with water and methanol. E2-imprinted monolith was smashed and the particles grounded. The grounded polymers were sieved and the fraction that fall in the size range of 5–10 ␮m were used throughout the study. Non-imprinted particles (NIP) were prepared, as control polymer, in the same manner as that previously described, without using E2 as template. 2.4. Production of PHEMA cryogel with MIP/NIP particles Production of the PHEMA cryogel with embedded MIP particles is described below. Briefly, monomers (1.6 mL HEMA and 0.3 g N,Nmethylene-bis(acrylamide) (MBAAm) were dissolved in deionized water (5 mL) and the mixture was degassed under vacuum for about 5 min to eliminate soluble oxygen. Total concentration of monomers was 12% (w/v). The cryogel was produced by free radical polymerization initiated by TEMED and APS. After adding APS (25 ␮L, 1% (w/v) of the total monomers) the solution was cooled in an ice bath for 2–3 min. Then, TEMED (20 mg, 1% (w/v) of the

total monomers) was added and the reaction mixture was stirred for 1 min. In this step, the MIP particles (150 mg) were mixed with the polymerization mixture. Then, the reaction mixture was poured between two glass plates separated with 1.5 mm thick spacers. The polymerization solution in the plates was frozen at −16 ◦ C for 24 h and then thawed at room temperature. The resulting cryogel sheets were cut into circular pieces (2 cm diameter) with a perforator. All of the experiments were performed with a 2 cm diameter column including three pieces of the cryogel membrane sheet. After washing with 200 mL of water, elution solution (acetonitrile: methanol (70:30 (v/v)) was passed through the PHEMA/MIP composite cryogel at room temperature for 3 h. This procedure was repeated until no E2 leakage was observed from the polymeric structure to the wash solution. The E2 free composite cryogel was washed with ethanol and water at room temperature for 12 h and the cryogels were stored in buffer containing 0.02% sodium azide at 4 ◦ C until use. 2.5. Characterization of cryogel Water uptake ratio (S) of the PHEMA/MIP composite cryogel was determined in distilled water. The experiment was conducted as follows: initially dry cryogel was carefully weighed before being placed in a 40 mL vial containing distilled water. The vial was put into an isothermal water bath with a fixed temperature (25 ◦ C) for 2 h. The sample was taken out from the water, wiped using a filter paper, and weighed. In different time intervals the weight of the cryogel was recorded. After 24 h the final weight of cryogel was recorded. The water uptake ratio of the cryogel was calculated as S=

 (W − W )  s 0 W0

× 100

(1)

where W0 and Ws are the weights of cryogel before and after uptake of water, respectively. The gelation yield was determined as follows: the swollen cryogel sample (1 mL) was put in an oven at 60 ◦ C for drying. After drying till constant weight, the mass of the dried sample was determined (mdried ). The gel fraction yield was defined as Gelation yield =

m

dried

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 roughly estimated by weighing the sample (msqueezed gel ) after squeezing the free water from the swollen gel matrix, and then the porosity was calculated as Porosity (%) =

(mswollen gel − msqueezed gel ) mswollen gel

× 100%

(3)

All measurements were done in triplicate and the average values are presented. The flow-rate of water passing through the column was measured at the constant hydrostatic pressure equal to 100 cm water column corresponding to a pressure of ca. 0.01 MPa. At least three measurements were done for each sample. Porosity of the polymer sample was measured by the nitrogen sorption technique, performed on Flowsorb II, (Micromeritics Instrument Corporation, Norcross, USA). The specific surface area of composite cryogel in dry state was determined by multipoint Brunauer– Emmett–Teller (BET) apparatus (Quantachrome, Nova 2200E, 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 gas was performed at 21 ◦ C and desorption was performed at room temperature. Values obtained from desorption step was used for the specific surface area calculation.

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The morphology of a cross section of the cryogel was investigated by scanning electron microscope (SEM). The sample was fixed in 2.5% glutaraldehyde in 0.15 M sodium cacodylate buffer overnight, post-fixed in 1% osmium tetroxide for 1 h. Then the sample was dehydrated stepwise in ethanol and transferred to a critical point drier temperated to 10 ◦ C where the ethanol was changed for liquid carbon dioxide as transitional fluid. The temperature was then raised to 40 ◦ C and the pressure to ca. 100 bar. Liquid CO2 was transformed directly to gas uniformly throughout the whole sample without heat of vaporization or surface tension forces causing damage. Release of the pressure at a constant temperature of 4 ◦ C resulted in dried cryogel sample. Finally, it was coated with gold–palladium (40:60) and examined using a JEOL JSM 5600 scanning electron microscope (JEOL, JSM 5600, Tokyo, Japan). Fourier transform infrared (FTIR) spectra of MAT, E2 and the MAT–E2 complex, and MIP particles were obtained using a FTIR spectrophotometer (FTIR 8000 Series, Shimadzu, Japan). E2, MAT monomer, MAT–E2 complex, were dried in a vacuum oven. The dry sample (0.1 g) was thoroughly mixed with KBr (0.1 g, IR Grade, Merck, Germany), and pressed into a pellet form and the FTIR spectrum was then recorded. To evaluate the amount of MAT into the cryogel structure, it was subjected to elemental analysis using a Leco Elemental Analyzer (Model CHNS-932, USA). 2.6. Assay of steroids The detection of E2, cholesterol and stigmasterol was followed by using high performance liquid chromatography (HPLC) system (Ultimate-3000, Dionex, USA) equipped with LPG-3000 pump, WPS-3000 autosampler, TCC-3000 column department, PDA-3000 detector and column (Kromasil 100-5, Length/I.D; 150/4.6 mm). The acetonitrile–methanol–water mixture was used as mobile phase. Mobile phases A, B and C were water, methanol and acetonitrile respectively. The chromatographic separation was performed using a linear gradient at 0.5 mL/min flow rate. A linear gradient started from 10% B, 80% C and 10% A in 5 min, continued with increasing B from 10% to 65% and decreasing C from 80% to 35% in 1 min and finished in 20 min. 100 ␮L of steroid solution was injected into the column. The absorbance was monitored at 280 nm for E2, cholesterol and stigmasterol. The separation was performed at ambient temperature. The limit of detection (LOD) and limit of quantification (LOQ) were calculated from the following equations: s m s LOQ = 10 × m LOD = 3.3 ×

(4) (5)

where s is the standard deviation of response and m is the slope of the corresponding calibration curve. 2.7. E2 removal studies from aqueous media The E2 adsorption studies were carried out in surface water mimicking medium with a continuous system, in a recirculation system equipped with a water jacket for temperature control. The MIP composite cryogel was washed with 30 mL of water for 30 min. Then, the E2 solution was pumped through the column under recirculation for 2 h. The adsorption was followed by using spectrophotometer (UV-1601, Shimadzu, Japan) the absorbance was monitored at 280 nm for E2. Effects of E2 concentration, flow-rate on the adsorption amount were studied. The effect of the initial concentration of E2 on adsorption capacity was studied by changing the concentration of E2 between 50 and 100 ␮g/L by using large volume (2 mL) injection with optional loop and 5–50 mg/L. The effect

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of flow rate on adsorption capacity was investigated at different flow rates in the range of 0.5–4.0 mL/min when pumped through the cryogel column under recirculation for 2.0 h with 30 mg/L of E2 solution in methanol–water (10:90 (v/v)). The amount of E2 adsorption per unit mass of dry cryogel was calculated using the mass balance. Each data collected is average of three determinations. 2.8. Selectivity experiments In order to show the specificity of PHEMA/MIP composite cryogel, competitive adsorption of cholesterol (MW: 386 g/mol) and stigmasterol (MW: 412.7 g/mol) was also studied. E2 aqueous solution was overloaded with cholesterol and stigmasterol and applied on PHEMA/MIP column. E2, cholesterol and stigmasterol were added in methanol:water (10:90 (v/v)) solution and sonicated for 10 min at room temperature. After attaining adsorption equilibrium, the concentrations of cholesterol and stigmasterol in the remaining solution were measured by the same HPLC system explained in Section 2.6. The distribution coefficient (Kd ) for cholesterol and stigmasterol with respect to E2 was calculated by Kd =

 (C − C )  i f Cf

×

V m

(6)

where Kd represents the distribution coefficient (mL/g); Ci and Cf are initial and final concentrations of E2 (mg/mL), respectively. V is the volume of solution (mL) and m is the weight of the cryogel column (g). The selectivity coefficient (k) for the binding of E2 in the presence of other competitive molecules can be obtained from binding data according to k=

Kd Kd

(7)

where k is the selectivity coefficient and X represents cholesterol or stigmasterol. A comparison of the k values of the PHEMA/MIP cryogel with those cholesterol or stigmasterol allows an estimation of the effect of imprinting on selectivity. A relative selectivity coefficient 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/MIP composite cryogel, the adsorption–elution cycle was repeated 10 times using the same PHEMA/MIP composite cryogel in a continuous experimental set-up. For sterilization after one adsorption–elution cycle, the cryogel was washed with 50 mM NaOH solution for 30 min. After this procedure, cryogel was washed with distilled water for 30 min. Elution of E2 was studied with acetonitrile: methanol (70:30 (v/v)) solution. PHEMA/MIP composite cryogel was contacted with elution medium for 2 h at room temperature. The final E2 concentration in the elution medium was measured as described above. The elution ratio was calculated from the amount of E2 adsorbed on the cryogel and the final E2 concentration in the elution medium. 3. Results and discussion 3.1. Characterization of composite cryogels Table 1 presents the swelling properties and linear flow resistance (at hydrostatic pressure, ca. 0.01 mPa) of both PHEMA/MIP and PHEMA/NIP cryogels, respectively. Cryogels have supermacropores, as a consequence of this; the flow rate through the gel

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1822 Table 1 Swelling properties and flow resistance of cryogels. Polymer

Monomer concentration (%)

Gel yield (%)

Swelling ratio (%)

Swelling degree (g H2 O/g polymer)

Macroporosity (%)

Flow rate (cm h−1 )

PHEMA/MIP PHEMA/NIP PHEMA

10 10 10

90.2 91 94.2

87 85 90.4

8.1 8.2 9.2

68 65 78

554 548 765

matrix is high. PHEMA/MIP showed the maximum flow rate at 554 cm h−1 and NIP showed a flow rate at 548 cm h−1 . Both MIP, NIP cryogels were produced with high gelation yield (about 90%) and had similar swelling properties with a swelling degree of about 8.0 g H2 O/g polymer as compared to cryogels prepared by Baydemir et al. (about 6.1 g H2 O/g polymer) [30] and Derazshamshir et al. (about 10 g H2 O/g polymer) [31].

FTIR spectra of the MAT monomer, E2, and MAT–E2 complex are shown in Fig. 1A. FTIR spectrum of pure E2 has characteristic peaks, which include H bonded O–H at 3438 and 3222 cm−1 and strong hydrocarbon peaks around 2900 cm−1 . Stretching vibration bands are at 1610 and 927 cm−1 . FTIR spectrum of MAT has the characteristic stretching vibration of amide I and amide II absorption bands at 1653 cm−1 and 1516 cm−1 , a carbonyl band at 1733 cm−1 ,

Fig. 1. FTIR spectra of (A) E2, MAT monomer and MAT–E2 complex. (B) PHEMA/MIP polymer and MIP particles.

I˙ . Koc¸ et al. / Journal of Hazardous Materials 192 (2011) 1819–1826 Table 2 Specific surface area of the polymers (m2 /g). Specific surface area (m2 /g) PHEMA PHEMA/MIP PHEMA/NIP

25.0 123.2 115.3

and an aromatic C–H band at 809 cm−1 . The functional monomer MAT is expected to interact with E2 through hydrogen bonds and hydrophobic interactions through an aromatic ring. Furthermore, the aromatic peak at 801 cm−1 of MAT monomer shifts upfield to 809 cm−1 , because of hydrophobic interactions. The E2–MAT complex has also characteristic peaks of H bonded O–H bond at 3559 cm−1 , amide I at 1654 cm−1 and amide II at 1515 cm−1 .The FTIR spectrum of the MAT monomer and of the E2–MAT complex are almost same, without shifts in wave numbers of related peaks, it can be concluded that the characteristic peaks of MAT monomer cover E2’s characteristic peaks in the E2–MAT complex spectrum. FTIR spectrum of MIP particles and PHEMA/MIP polymer is shown in Fig. 1B. All include H bonded O–H at around 3430 and 3000 cm−1 and strong hydrocarbon peaks around 2950 cm−1 . The SEM images of the internal structures of the MIP particles embedded PHEMA/MIP composite cryogel are shown in Fig. 2. PHEMA/MIP composite cryogel has continuous interconnected pores (10–100 ␮m in diameter) that provide high flow rates through the channels. SEM images showed that the MIP particles were uniformly distributed into the PHEMA cryogel network. Pore size of the matrix is much larger than the size of the E2 molecules, allowing them to enter easily through the pores of the convective flow of the water through the pores; the mass transfer resistance is practically negligible. The specific surface area of the PHEMA/MIP composite cryogel was determined to be 123.2 m2 /g polymer and the specific surface area of the PHEMA/NIP composite cryogel column was determined to be 115.3 m2 /g (Table 2). The incorporations of the MAT for MIP and NIP particles were determined to be 78 ␮mol/g and 72 ␮mol/g polymer, respectively, using nitrogen stoichiometry. Note that HEMA and other polymerization ingredients do not contain nitrogen. This nitrogen amount, determined by elemental analysis, comes from only incorporated MAT groups into the polymeric structure. 3.2. Removal of template The acetonitrile–water solution was passed through the PHEMA/MIP composite cryogel for the removal of the template from 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.

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

Linearity range (␮g mL−1 )

Slope

Intercept

S.D. of slope

S.D. of intercept

Correlation coefficient

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

Fig. 4. Effect of equilibrium E2 concentration on adsorption amount: Flow-rate: 0.5 mL/min; T: 25 ◦ C, Time: 2 h.

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

shows the decrease of amount of template E2 released from the PHEMA/MIP polymer. The peaks labeled as 1–6 shows the amount of E2 released from the PHEMA/MIP cavities. After 6 elution procedure, no significant E2 peak was observed which means that almost all amount of E2 was removed from PHEMA/MIP composite cryogel. The E2 concentration was detected by HPLC. The calibration data was constructed by the plotting of absorbance versus standard concentration (Table 3). The LOD for E2 was determined as 0.768 ␮g mL−1 .

3.4. Adsorption isotherm

3.3. E2 adsorption studies 3.3.1. Effect of equilibrium concentration of E2 Fig. 4 shows the equilibrium concentration of E2 dependence of the adsorbed amount of E2 onto the PHEMA/MIP composite cryogel. The amount of adsorbed E2 increased with increasing E2 initial concentration, and a saturation value is achieved at E2 concentration of 30 mg/L which represents saturation of the accessible binding cavities on the PHEMA/MIP composite cryogel. Maximum adsorption capacity was 5.32 mg/g polymer. The MIP particle embedded composite cryogel play the significant role for binding E2 molecules, as a result of that PHEMA/MIP composite cryogel showed a 7 times higher capacity to adsorb the E2 in water than the PHEMA cryogel with NIP particles (0.8 mg/g polymer) (Fig. 4).

3.3.2. Effect of flow rate The amounts of adsorbed E2 at different flow-rates are given in Fig. 5. Results show that the E2 adsorption capacity onto the PHEMA/MIP composite cryogel decreased when the flowrate through the column increased. The amount of adsorbed E2 decreased significantly from 5.32 mg/g to 3.2 mg/g polymer with the increase of the flow-rate from 0.5 mL/min to 4.0 mL/min. This is due to decrease in contact time between the E2 molecules and the composite cryogel at higher flow-rates. These results are also in agreement with those referred to the literature [32]. When the flow-rate decreases, the contact time in the column is longer. Thus, E2 molecules have more time to recognize the E2 molecular cavities in embedded MIP particles in the PHEMA cryogel structure and to enter to the molecular cavities; hence a higher adsorption amount is obtained.

Modeling of the equilibrium adsorption data has been done using the Langmuir and Freundlich isotherms [33]. A good fit is the 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 Langmuir adsorption equilibrium constant, which indicates monolayer adsorption. The other well-known isotherm, which is frequently used to describe adsorption behavior, is the Freundlich isotherm (8): 1/n

q = KF Ceq

(10)

where KF is the Freundlich adsorption constant (mg/mL), Ceq is the equilibrium E2 concentration (mg/mL), and n is the Freundlich exponent which represents the heterogeneity of the system. The Freundlich isotherm describes reversible adsorption and is not restricted to the formation of the monolayer. A more homogeneous system will have n value approaching unity while a more heterogeneous system will have an n value approaching zero [34]. In Table 4, the experimental adsorption behavior was compared with Langmuir and Freundlich adsorption isotherms. The experimental data tend to be better fitted with Langmuir rather than Freundlich isotherm, since the correlation coefficient (R2 ) was high (0.98). The maximum amount of adsorption (5.32 mg/g) obtained from experimental results is also close to the calculated Langmuir adsorption capacity (5.93 mg/g). The Langmuir and Freundlich adsorption constants with the correlation coefficients are given in Table 4. It can be concluded that the adsorption of E2 onto PHEMAH/MIP cryogel is a monolayer adsorption. The factors affecting adsorption mechanism, such as mass transfer and the binding itself were investigated using two different kinetic models. The pseudo-first order and pseudo-second order equations can be used in this case assuming that the measured concentrations are equal to adsorbent surface concentration. The first-order rate expression of Lagergren [35] is one of the most

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Table 4 Langmuir and Freundlich adsorption isotherm constants. Experimental

Langmuir constants

qex (mg/L)

qmax (mg/g)

b (mL/mg)

R2

KF

Freundlich constants n

R2

5.32

5.93

0.00

0.98

36.23

2.3

0.75

Table 5 The first and second order kinetic constants for PHEMAH/MIP. Initial concentration (mg/L)

30

Experimental

First-order kinetic

qeq (mg/g)

k1 (1/min)

qeq (mg/g)

R2

Second-order kinetic k2 (1/min)

qeq (mg/g)

R2

5.32

0.113

6.828

0.8413

0.00724

5.36

0.9498

Fig. 6. Chemical structures of competitor molecules.

widely used for the adsorption of solute from a liquid solution, Eq. (11): log(qe − qt ) =

log(qe ) − (k1 t) 2.303

(11)

where qe is the experimental amount of E2 adsorbed at equilibrium (mg/g); qt is the amount of E2 adsorbed at time t (mg/g); k1 is the rate constant of the pseudo-first order adsorption (min−1 ). A linear dependence of log(qe − qt ) on t suggests the applicability of this kinetic model. Also, in many cases, the pseudo-first order equation of Lagergren does not fit well to the whole range of contact time and is generally applicable over the initial stage of the adsorption processes. The pseudo-second order kinetic model is expressed by Eq. (12):

t qt



=

1 k2 q2eq

  +

1 qeq



t

(12)

where k2 is the rate constant of the pseudo-second order adsorption (g/mL min). If the pseudo-second order kinetics is applicable, the plot of t/q versus t should be linear. The pseudo-second order kinetic model is favorable, when the adsorption behavior over the whole range of adsorption is in agreement with chemical adsorption being the rate controlling step [36]. A comparison of the experimental adsorption capacity and the theoretical values is presented in Table 5. The correlation coefficient for the linear plot of −log(qeq − qt ) versus t for the pseudo-first order equation is lower than the correlation coefficient for the pseudo-second order equation suggesting that the pseudo-second order adsorption mechanism is predominant and that the overall rate of the E2 adsorption process appeared to be controlled by binding kinetics.

tures of E2, cholesterol and stigmasterol. Molecular weight of E2 is 272.4 g/mol while that of cholesterol is 386 g/mol and that of stigmasterol is 412.7 g/mol. Table 6 summarizes the Kd , k and k values in selectivity studies. Distribution coefficient (Kd ) value for the PHEMA/MIP composite cryogel is higher than that of the control PHEMA/NIP cryogel. Kd value showed an increase for E2 while it tends to decrease for the competitor molecules, cholesterol and stigmasterol. The relative selectivity coefficient is an indicator to express binding affinity of recognition sites to the imprinted E2 molecules [14,31]. The results show that relative selectivity coefficients of PHEMA/MIP composite cryogel for E2/cholesterol and E2/stigmasterol were 7.6 and 85.8 times greater than the PHEMANIP cryogel, respectively. Selectivity is dependent on the shape and size memories of the imprinted cavities. The competitor molecules were less adsorbed by the PHEMA/MIP composite cryogel due to linear chains in their chemical structures makes them more hydrophobic than E2. E2 has two hydroxyl groups so it constitutes strong hydrogen bonding through hydroxyl group on specific monomer tyrosine in PHEMAT/MIP cavities. Nevertheless, the binding specificity for E2 from PHEMA/MIP composite cryogel was sufficient for the recognition of E2 from other compounds. Fig. 7 shows the adsorbed template and competitive molecules both in PHEMA/MIP composite cryogel and PHEMA cryogel in mg/g polymer.

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

NIP

3.5. Selectivity studies The specificity of PHEMA/MIP cryogel to recognition of E2 is investigated by using cholesterol and stigmasterol; molecules have similar chemical structure with E2. Fig. 6 shows the chemical struc-

Polymer

E2 Cholesterol Stigmasterol

MIP

Kd

k

Kd

k

k

89.2 41.6 208.3

2.1 0.4

851 52 23

16.34 36.76

7.6 85.8

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the monolithic cryogel column. The E2 removal from water with competitor molecules (E2 concentration 30 mg/L) was found as 5.32 mg/g polymer with a considerably high removal ratio (88%). There is no significant decrease in adsorption capacity after several adsorption–desorption cycles. The results presented here demonstrate that the PHEMAH/MIP composite cryogels can be used for the recognition and selective removal of E2 molecules from water with a high E2 removal capacity. References

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

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

3.6. Elution and repeated use In order to show the stability and reusability of the PHEMA/MIP composite cryogel, the adsorption–elution cycle was repeated 10 times using the same PHEMA/MIP composite cryogel in a continuous experimental set-up. For sterilization after one adsorption–elution cycle, the cryogel was washed with 50 mM NaOH solution for 30 min. After this procedure, cryogel was washed with distilled water for 30 min. At the end of 10 adsorption–elution cycle, there was no remarkable decrease in the E2 adsorption capacity (Fig. 8) and the percent removal ratio of E2 was found as 88%. As seen here that the PHEMA/MIP composite cryogel is very stable, and maintain their adsorption capacity at almost constant value of 96.3%. 4. Conclusion In this study PHEMAH/MIP composite cryogels combined high flow path through interconnected supermacroporous structures of cryogels with high selectivity of the MIP particles PHEMAH/MIP composite cryogels were produced and high selectivity for E2 was demonstrated. The PHEMAH/MIP composite cryogels were prepared using 5–10 ␮m MIP particles. The incorporation of the MIP particles not only enhances the surface area of the PHEMA cryogel (about 5 fold) but also increase the mechanical stability of

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