Reactive & Functional Polymers 71 (2011) 402–408

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Reactive & Functional Polymers

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Physico-chemical characterization of flavonol molecularly imprinted polymers

Luis E. Gómez-Pineda a,d, Georgina E. Pina-Luis a,⇑, Ángeles Cuán b, Josefa A. García-Calzón c,Marta E. Díaz-García c

a Centro de Graduados e Investigación, Instituto Tecnológico de Tijuana, AP 1166, Tijuana, BC 22500, Mexicob Departamento de Materiales, Universidad Autónoma Metropolitana Unidad Azcapotzalco, Av. San Pablo 180, Col. Reynosa Tamaulipas, 02200 México DF, Mexicoc Departamento de Química Física y Analítica, Universidad de Oviedo. C/Julián Clavería 8, Oviedo 33006, Españad Centro de Ingeniería y Tecnología, Universidad Autónoma de Baja California, Blvd. Universitario #1000, Unidad Valle de las Palmas, Tijuana, BC, Mexico

a r t i c l e i n f o

Article history:Received 10 August 2010Received in revised form 19 December 2010Accepted 26 December 2010Available online 9 January 2011

Keywords:Molecularly imprinted polymerHeterogeneityAdsorption isothermsDFT

1381-5148/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.reactfunctpolym.2010.12.013

⇑ Corresponding author.E-mail address: gpinaluis@tectijuana.mx (G.E. Pina

a b s t r a c t

Along the last decade considerable interest has been focused in deciphering the binding recognition pro-cess and the heterogeneity nature of binding sites in molecularly imprinted polymers (MIPs). DensityFunctional Theory (DFT) was employed at B3LYP/6-31+G�� level of theory, to investigate the intermolec-ular interactions between the template flavonol and the functional monomer (methacrylic acid). The sol-vent effect energy stabilization of the monomer and template in different solvents was investigated byusing the polarizable continuum method (PCM). The MIP against flavonol prepared using methacrylicacid as functional monomer was then characterized in terms of specific bindings by using Scatchard plotanalysis. The rebinding properties were analyzed by equilibrium/rebinding batch experiments using theLangmuir, Bi-Langmuir, Freundlich, Langmuir-Freundlich, Jovanovich, Bi-Jovanovich and Jovanovich-Fre-undlich isotherms. The imprinted polymer was also characterized by continuous flow experiments, SEMand BET surface area measurements. Parallel studies were carried out with the non-imprinted polymer ascontrol material.

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1. Introduction

Molecular imprinting is a powerful technology for the synthesisof polymeric materials bearing biomimetic binding sites [1–3].Molecularly imprinted polymers are synthesized by copolymeriza-tion of functional monomer(s) and cross-linker(s) in the presenceof the template molecule to be recognized. A commonly usedmethod to imprint a molecular mark in a polymeric material isbased on non-covalent self-assembly of the template moleculewith functional monomers prior to polymerization. Free radicalpolymerization with a cross-linking monomer stabilizes the tem-plate-functional monomer complexes, resulting in a mechanicallyand thermally stable polymer. After extractive removal of the tem-plate, the remaining molecularly imprinted polymer (MIP) con-tains 3-dimensional binding cavities that rebind the templatedue to complementary in shape and functionality with it.

In the non-covalent approach the porogen (solvent) competeswith the functional monomers for interaction with the templatemolecule and so the binding sites formed have different conforma-tions and affinities [4]. The knowledge of the interaction mecha-

ll rights reserved.

-Luis).

nism between the template and the functional monomer is a keyissue in molecular imprinting as it may provide insights into thenature of binding sites heterogeneity. Besides, the stability and ex-tent of the monomer-template assembly in the pre-polymerizationmixture reveal the way to develop novel materials with high affin-ity and selectivity [5–7].

Several theoretical studies have proved that computational sim-ulations may be successfully applied in the rational design, evalu-ation and prediction of affinity and selectivity of MIPs [8–13]. In aprevious work [14], we have developed a molecularly imprintedpolymer against flavonol and used it as molecular recognitionmaterial in a flow-through approach, thus indirectly proving theexistence of imprinted binding sites. Here, we have extended thisinvestigation by further exploring structural and physicochemicalproperties of flavonol imprinted polymers using theoretical tech-niques and comparing them with experimental data. For studyingthe pre-polymerization complex, methacrylic acid (MAA) and dif-ferent porogens were evaluated to perform the synthesis. Thebinding energy of the functional monomer-template complexwas calculated using a Density Functional Theory at B3LYP/6-31+G�� level. The quantum chemical calculations were carriedout at vacuum conditions and taking into account the solvent ef-fect, implemented in Gaussian package [15]. The solvent effectwas included using the polarizable continuum method (PCM)

L.E. Gómez-Pineda et al. / Reactive & Functional Polymers 71 (2011) 402–408 403

[16–18]. Also, to better understand adsorption of flavonol, the dy-namic process of adsorption, the textural and morphological fea-tures of MAA-based imprinted polymer and different isothermmodels were assayed to evaluate the recognition process offlavonol.

2. Experimental

2.1. Reagents and instruments

3-Hydroxyflavone (flavonol) was purchased from Aldrich (Mil-waukee, WI), ethylene glycol dimethacrylate (EGDMA), metha-crylic acid (MAA) and a,a’-azoisobutyronitrile (AIBN) wereproducts of Fluka (Buchs, Switzerland). All solvents used were ofanalytical grade. Fluorescence measurements were performedusing a Perkin–Elmer (LS-50B) Luminescence Spectrometer usinga quartz cell of 10 mm light path. The excitation and emissionwavelengths were set at 340 and 525 nm, respectively. Imprintedand non-imprinted polymers were examined with a Ziess DSM942 scanning electron microscope (SEM).

2.2. Polymer preparation

Imprinted polymers having flavonol recognition sites were pre-pared according to a simple method [14] using MAA, that was pro-posed to form favorable non-covalent interactions with thetemplate, EGDMA as cross-linking agent that served as themechanical support and chloroform as porogenic solvent (Fig. 1).In order to synthesize the MIPs 9.0 mg (0.038 mmol) of flavonol,0.269 mL (1.423 mmol) of EGDMA, 0.026 mL (0.302 mmol) ofMAA, 1.2 mL of chloroform and 3.0 mg (0.019 mmol) of AIBN asradical initiator were mixed in a glass vial. The oxygen of the solu-tion was removed by bubbling N2 for 5 min. The vial was sealedand heated in an oven at 70 �C for thermal initiation of AIBN. Thecontrol blank polymer was prepared using an identical procedurein the absence of the template. After 14 h heating, the resultingpolymers were mechanically crushed and sieved (particles ofdiameters ranging between 0.16 and 0.08 lm). To remove the tem-plate molecule, the polymers particles were Soxhlet rinsed usingmethanol (8 h, 40 cycles).

2.3. Scatchard analysis and adsorption isotherms

Equilibrium isotherm data were obtained in a batch wise ap-proach. Pre-weighed amounts of cleaned imprinted polymer andblank polymer (11 mg) were contacted with hexane solutions(2 mL) of flavonol of known concentrations (9.0 � 10�6–1.0 � 10�4 M). The resulting suspensions were shaken for 12 h atroom temperature. Then the polymers were rapidly removed by fil-tration and flavonol in the resulting solution was analyzed by fluo-rescence, the excitation and emission wavelengths being 340 and

Fig. 1. Schematic representation of p

525 nm, respectively. The concentration of flavonol bound to thepolymer, B (mol L�1), was calculated by subtraction of the concen-tration of free flavonol, [F] (mol L�1), from the initial flavonol con-centration. [F] was determined as an average value of threemeasurements. Scatchard analysis was provided by the Scatchardequation:

B=½F� ¼ ½Bmax � B�Ka ðIÞ

where Ka (L mol�1) is the association constant and Bmax (mol L�1) isthe apparent maximum number of binding sites. Batch rebindingexperiments and Scatchard analysis were done in a similar wayfor the corresponding blank polymer. The most adequate adsorptionisotherm model was selected using a modified Fisher test, accordingto which the model that better correlated the data was the one thatexhibited the highest value of the F-value [19].

2.4. Textural and structural characterization of imprinted polymers

Textural characterization of the imprinted and control polymerswas carried out by measuring the N2 adsorption isotherms at 77 Kin a Micromeritics ASAP 2000 apparatus that allowed to control theliquid nitrogen level (within ±0.2 mm) and the dosing volumedeviation (<0.5%). The apparatus was equipped with pneumaticallyoperated control valves (maintained at 25.00 ± 0.05 �C) and equi-librium pressures were measured with an accuracy of ±0.15% ofreading. Before the experiments, the samples were out-gassed un-der vacuum at 383 K overnight. The Brunauer–Emmett–Teller(BET) surface area, total pore volume (VT) and average pore sizewere calculated. Samples, prepared in three different batches, wererun in duplicate. Reproducibility of textural analyses was in the95% confidence level within the range of relative pressures studied.Imprinted (and non-imprinted) polymers were dispersed on agraphite adhesive tab placed on an aluminium stub, coated witha thin layer of gold and examined with a Ziess DSM 942 scanningelectron microscope (SEM). Pictures of each sample were takenat 500� magnification and scanned directly to achieve the SEMimage.

2.5. Adsorption dynamics

Eleven milligram of imprinted polymer was filled in a glass col-umn of 5 mm of diameter and 0.6 cm of bed height. A hexane fla-vonol solution (6 � 10�5 mol L�1) was allowed to flow graduallythrough the column at a rate of 0.22 mL min�1. The effluent was al-lowed to enter into a flow-cell positioned into a spectrofluorimeterto monitor the flavonol fluorescence at 525 nm while excitationwas set at 340 nm. The dynamics adsorption curve was continu-ously registered. Breakthrough took place when a concentrationof flavonol (C, mol L�1) corresponding to a percentage of the initialconcentration (C0, mol L�1) in the feed reservoir was detected inthe effluent. The work exchange capacity (WEC) expressed the to-

olymer imprinting for flavonol.

Fig. 2. Optimized configurations of: (a) flavonol, (b) methacrylic acid and (c) 1:1flavonol-MAA assembly.

Table 1DEsolvent (kcal mol�1) for flavonol and MAA in different solvents.

Environment DEsolvent of flavonol DEsolvent of MAA

Vacuum – –Chloroform �7.3983505 �6.8162188THF �8.5461735 �7.8431225Acetonitrile �10.7230965 �9.5898315

MAA: methacrylic acid; THF: tetrahydrofuran.

404 L.E. Gómez-Pineda et al. / Reactive & Functional Polymers 71 (2011) 402–408

tal amount of flavonol adsorbed by the polymer before break-through occurred and when expressed by mass unit of the materialwas given by the equation:

WEC ¼ tRC0

mðIIÞ

in which C0 (mol L�1) was the flavonol concentration in the feedcarrier, m (g) was the mass of the polymer used and tR (min) wasthe breakthrough time, corresponding to a flavonol concentrationof 3 � 10�6 mol L�1 (5% of C0).

2.6. Molecular simulation

All calculations have been carried out using Gaussian 03 soft-ware [15] using density functional theory (DFT) at the B3LYP/6-31+G�� level. First, the optimized conformations of flavonol (FL)and MAA and related energies were obtained. Then, the bindingenergy, DEbinding (kcal mol�1), was calculated through the equation:

DEbinding ¼ EðcomplexÞ � EððFLÞ � EðmonomerÞÞ ðIIIÞ

Gas phase predictions are appropriate for many purposes; how-ever, they are unsuitable to explain the characteristics of polarmolecules in solution. In order to consider the condensed-phase ef-fects, a self-consistent reaction field model proposed for quantumchemical computations on solvated molecules was employed [20–23]. The solvent energy, DEsolvent (kcal mol�1), was calculatedthrough the equation as follows:

DEsolvent ¼ EðsolutionÞ � EðvacuumÞ ðIVÞ

where E(vacuum) and E(solution) are the interaction energy in thecondition of gas and taking into account the solvent effect, respec-tively. Thus, a higher DEsolvent stabilization indicates a strongeraffinity of flavonol or MAA to the solvent, which hinders access tothe template molecule by the monomer, leads to a lower adsorptionselectivity of the MIP.

3. Results and discussion

3.1. Molecular simulation

Geometries of isolated reactants, flavonol and MAA, were ini-tially optimized, finding that the most stable conformation of fla-vonol shows an intramolecular hydrogen bond between thehydroxyl and carbonyl groups. The optimized geometry of themost stable complex between flavonol and MAA showed that thehydroxyl and carbonyl groups from flavonol could interact molec-ularly with the hydroxyl and carbonyl groups from the MAA mono-mer through hydrogen bond donor and hydrogen bond acceptor.The optimized conformations of flavonol, the functional monomerMAA and flavonol-MAA assembly are shown in Fig. 2.

To quantitatively evaluate the stabilization of the interaction be-tween the template molecule and the monomer, the binding energy,DEbinding, was calculated. In vacuum conditions theDEbinding of stabil-ization for the flavonol complex formation was about12.13 kcal mol�1. From these results, it was clear that not onlyattractive interactions between flavonol and MAA, through the hy-droxyl and carbonyl groups, but also their spatial orientation (ste-reoelectronic complementarity) play an important role in thestabilization of pre-polymerization complexes for preparationof selective MIPs against flavonol, as has been reported previously[24].

The solvent had an important role in the interaction betweenthe monomer and template molecule. The DEsolvent of flavonoland MAA in different solvents were calculated according Eq. [IV]to characterize the strength of their respective interaction with sol-

vents. The values reported in Table 1 were estimated in differentsolvent models by the Polarizable Continuum Method (PCM)[16–18].

The DEsolvent stabilization of flavonol in acetonitrile is the larg-est, indicating the strongest interaction between flavonol and ace-tonitrile. The DEsolvent stabilization of flavonol in chloroform wasthe smallest, while the THF had intermediate interaction strengthof stabilization with flavonol as it is compared with chloroformand acetonitrile. The same calculation procedure was conductedfor MAA and results of the calculation are also summarized in Ta-ble 1. The magnitude of DEsolvent of MAA in the different solventsshowed the same order of energy stabilization, i.e., DEsolvent (aceto-nitrile) >DEsolventðTHFÞ > DEsolvent (chloroform). Based on the resultsin Table 1 it was expected that chloroform had the lowest affinityfor both the template, flavonol, and the monomer, MAA. This lowaffinity acted to enhance the interaction between flavonol andMAA, which was required in the formation of the flavonol–MAAcomplex prior to the initiation of polymerization. Acetonitrile, onthe other hand, had the highest deleterious effect on the formationof the flavonol-MAA complex, as indicated by the highest DEsolvent

stabilization value.

3.2. Preparation of molecularly imprinted polymers

The interactions between the template and imprinted polymerusually depend heavily on the presence of hydrogen bonds duringthe polymerization process. As water molecules tend to obstructthat interactions and recede binding of the target with the

Fig. 3. Flow-injection set-up for optical sensing of flavonol using a MIP packed flowcell.

L.E. Gómez-Pineda et al. / Reactive & Functional Polymers 71 (2011) 402–408 405

monomer, most MIPs are synthesized and used in organic solvents[25]. Taking into account that flavonol was insoluble in polar sol-vents; porogens of different polarity were assayed in the synthesisof molecularly imprinted polymers (as well as the control materi-als): methanol, acetonitrile, THF and chloroform. Once the materi-als were grounded and flavonol was removed, they were assayedfor flavonol rebinding using a flow-injection set-up, in which thepolymers were packed into a conventional flow-cell (see Fig. 3).

In this dynamic system, flavonol was injected in a carrier con-sisting in a mixture of n-hexane and the solvent used for synthesis

Fig. 4. SEM images of MAA-based imprinted polymers for flavonol synthesized in

Fig. 5. N2 adsorption isotherms of: (a) flavon

(n-hexane:porogen solvent volume ratio 90:10). n-hexane is apoorly hydrogen bonding solvent [26]; consequently, its abilityto compete for the hydrogen bonding sites of flavonol or the poly-mer binding sites should be reduced, thus favouring flavonolrebinding. On the other hand, the porogen was used as the modifierto achieve flavonol retention in the MIP within a practical timewindow. Results demonstrated that the imprinted polymer pre-pared using chloroform as porogen exhibited specific flavonolbinding sites and that the signal for flavonol bound to the polymerincreased in the order MIPmethanol < MIPacetonitrile < MIPTHF < MIPchlo-

roform, following the decreasing ET(30) polarity values [27] of thesolvents: 55.4(methanol) > 45.6(acetonitrile) > 37.4(THF) � 39.1(chloroform). In Fig. 4 the SEM images of the different imprintedpolymers are shown. As can be seen, the morphology of the mate-rials was solvent-dependent: the surface of flavonol-MIPs preparedin methanol was composed of aggregates of packed primary parti-cles in the size range 100–200 nm (see Fig. 4a) while the surface ofthose prepared in THF or chloroform (Fig. 4c and d) were morespongy and comprised of numerous microspheres smaller thanthose of polymers prepared in methanol or acetonitrile, suggestinga larger surface area for these polymers than for the other two.

3.3. N2 adsorption

Fig. 5 depicts the N2 adsorption–desorption isotherms for theimprinted polymer with its corresponding control. Detailed char-acteristics of the pore structure of the imprinted and non-im-

different solvents and the corresponding flow-injection response for flavonol.

ol-MIP and (b) non-imprinted polymer.

Table 2Textural parameters of imprinted and non-imprinted polymers, obtained from the N2

adsorption data at 77 K.

Parameter Imprinted polymer Non-imprinted polymer

Surface area (m2/g) 227.24 207.01Vpores (cm3/g) 0.5449 0.6416Average size pore (Å) 95.92 113.23

Fig. 6. Scatchard plots for flavonol-MIP and its corresponding NIP.

Table 3Adsorption isotherm models.

Freundlich B = aFm (1)Langmuir B ¼ NKF

1þKF(2a)

Bi-Langmuir B ¼ N1 K1 F1þK1 F þ

N2K2 F1þK2F

(2b)

Freundlich-Langmuir B ¼ NKFm

1þKFm (2c)

Jovanovic B = N(1 � e�KF) (3a)Bi-Jovanovic B ¼ N1ð1� e�K1F Þ þ N2ð1� e�K2 F Þ (3b)Freundlich-Jovanovic B ¼ Nð1� e�KFm Þ (3c)

B: concentration of flavonol adsorbed (mol L�1); F: concentration of flavonol notadsorbed (mol L�1); K: affinity constant (L mol�1); N: binding site density (mol L�1);m: heterogeneity parameter.

406 L.E. Gómez-Pineda et al. / Reactive & Functional Polymers 71 (2011) 402–408

printed polymers prepared using MAA are presented in Table 2. Itis worth noting that the BET surface area did not change signifi-cantly between the imprinted polymer and the control blank poly-mer. This result precluded the assumption that the porous featuresof the polymers were not dependent on the imprinted template.

3.4. Saturation experiments and Scatchard analysis

The interaction of a molecularly imprinted polymer with itstemplate can be described by a model of reversible binding, char-acterized by dissociation equilibrium constant. The key parameters

Table 4Fitting parameters obtained by non-linear regression for the different adsorption isotherm

Modelisotherm

Affinity constant, K(L mol�1)

Binding site density, N(mol L�1)

Binding(N � K)

L 4.3 � 10�4 1581 0.675Bi-L 1.03 � 10�4 10176 1.051

1.4 � 10�12 17,589 2.44 � 1J 2.0 � 10�4 2903Bi-J n.d. n.d. n.d.F – – 0.231L-F 2.43 � 10�6 94,938 0.231J-F n.d. n.d. n.d.

n.d.: fitting rejected as coefficient values were not significative.

can be deduced from concentration-dependent binding isotherms,and quantified most accurately by computer-assisted, non-linearregression analysis or fitted by the linear subtraction method.The earliest method of linearization of ligand-binding data accord-ing to Scatchard is still frequently employed. Scatchard analysis isan effortless and straightforward way to recognize multiple classesof binding sites and provides the most compact graphical presen-tation of binding data [4]. The hypothesis that all binding sitesare alike does not apply in most developed MIPs and usually Scat-chard plots fit to a curve which degree of curvature contains infor-mation about the affinity distribution of binding sites [28].Scatchard plots for the flavonol imprinted and the control polymerwere constructed by plotting the ratios of bound to free templateconcentration against the bound concentration for a constant massof polymer (11 mg). Results indicated that the recognition sites inthis imprinted polymer were not consistent in nature and, at least,two types of binding sites may exist there (Fig. 6), one of high affin-ity and other of low affinity. The binding constant of the high affin-ity sites was determined by the slope of the line running from they-intercept to the x-intercept, while that of the low affinity siteswas calculated by the slope of the line tangent to the curvatureat the x-intercept.

3.5. Adsorption isotherm models

To characterize the binding surface, isotherm parameters suchas the binding site density (Nt), the affinity constant (K) or the het-erogeneity parameter (m) were calculated by using the most suit-able adsorption isotherm model among the following: Langmuir(L), Bi-Langmuir (Bi-L), Jovanovic (J), Bi-Jovanovic (Bi-J), Freundlich(F), Langmuir-Freundlich (L-F), and Jovanovic-Freundlich (J-F) iso-therms (Table 3).

The models (L) and (J) offer a very simple point of view of thebinding behavior of polymers, with a unique affinity constant valueand no heterogeneity of the binding sites. On the contrary, themodels Bi-L and Bi-J, considering two different sorts of bindingsites with ‘‘high’’ and ‘‘low’’ affinity, show a more complex visionof the binding. The known heterogeneous binding behavior of thenon-covalent imprinted polymers can be represented by usingthe models L-F and J-F, in which the heterogeneity parameter m in-forms about the existence of a distribution of binding site classes.Experimental results are shown in Tables 4 and 5.

As can be seen, the higher values of the Fisher test for the Fre-undlich isotherm when compared to the Langmuir or the Jovanovicisotherms suggested that the Freundlich isotherm was an appro-priate model in estimating the flavonol adsorption parameters tothe imprinted polymer. The heterogeneity parameter, m, is a mea-sure of the ratio of high-to-low-affinity sites and varies from 0 to 1(values approaching to 0 indicate increasing heterogeneity,whereas values close to 1 correspond to a more energeticallyhomogeneous system). Both, the imprinted and the control poly-

s of imprinted polymer.

capacity Heterogeneityparameter, m

Regression coefficient,R2

Fisher test,F

– 0.996 1875– 0.943 33

0�8

– 0.996 1843– n.d. n.d.0.8996 0.998 32990.8993 0.998 1443n.d. n.d. n.d.

Table 5Fitting parameters obtained by non-linear regression for the different adsorption isotherms of non-imprinted polymer.

Modelisotherm

Affinity constant, K(L mol�1)

Binding site density, N(mol L�1)

Binding capacity(N � K)

Heterogeneityparameter, m

Regression coefficient,R2

Fisher test,F

L 4.73 � 10�4 1335 0.631 – 0.994 1367Bi-L 8.6 � 10�5 12,751 1.099 – 0.927 30

9.0 � 10�16 17,219 1.55 � 10�11

J 3 � 10�4 2449 – 0.993 1352Bi-J n.d. n.d. n.d. – n.d. n.d.F – – 0.227 0.9027 0.996 2185L-F 3.35 � 10�7 677,015 0.227 0.9027 0.996 971J-F n.d. n.d. n.d. n.d. n.d. n.d.

n.d.: fitting rejected as coefficient values were not significative.

Fig. 7. Affinity distributions for flavonol binding to MIP and to NIP calculated using the affinity distribution function plotted in terms of: (a) N(K) vs. ln(K) and (b) ln(N(K)) vs.ln(K).

Table 6Freundlich fitting parameters, weighted average affinity and number of sites forflavonol binding.

Parameter Imprinted polymer Non-imprinted polymer

a 0.231 0.227m 0.900 0.903R2 0.998 0.996NKmin�Kmax /mol L�1 1.02 � 10�5 9.12 � 10�6

Kn/L mol�1 2.7 � 104 2.8 � 104

Kn and NKmin�Kmax calculated in the range (ln(K) = 9.2–11.6 L mol�1).

0.0

0.2

0.4

0.6

0.8

1.0

600 1100 1600 2100 2600 3100

C/C

0

(a)

MIP

0.0

0.2

0.4

0.6

0.8

1.0

600 1100 1600 2100 2600 3100

C/C

0

Time (s)

(b)

NIP

WEC

WEC

Fig. 8. Breakthrough curves for: (a) flavonol-MIP and (b) NIP.

L.E. Gómez-Pineda et al. / Reactive & Functional Polymers 71 (2011) 402–408 407

mer have m values that are relatively constant, from 0.8993 to0.9027. Comparing the differences in values of heterogeneityparameter in the system under study, one can state that in the caseof the MIP the system showed relatively moderate heterogeneityeffects (m 6 0.9) in comparison to the control polymer for whichmuch homogeneity effects were found (m P 0.9).

The affinity distribution was plotted in terms of N(K) vs ln(K) orln(N(K)) vs. ln(K) using the data obtained from the Freundlich iso-therm, which gave the best fitting parameters. The affinity distri-bution in N(K) vs. ln(K) format resulted in an exponentiallydecreasing function for the imprinted polymer interactions, ascan be seen in Fig. 7a. For the highest association constant, theaffinity distribution function tended towards zero while it tendedtowards infinity for the lowest association constant. The formatln(N(K)) vs. ln(K) generated straight lines (Fig. 7b), suggesting thatthe isotherm model was consistent with the adsorption energy dis-tribution. In Table 6 the Freundlich fitting parameters aresummarized.

408 L.E. Gómez-Pineda et al. / Reactive & Functional Polymers 71 (2011) 402–408

3.6. Adsorption dynamics

When working sorption experiments with columns packed withthe imprinted polymer, the breakthrough curves are very useful. Aplot of effluent template concentration (or a parameter relatedwith it) vs. time usually yields as S-shaped curve, at which thetemplate concentration reaches its maximum allowable value isreferred to as a breakthrough curve. The point where the effluenttemplate concentration reached 5% of its influent value was takenas the breakthrough point in this work. As shown on Fig. 8 the runfor the molecularly imprinted polymer has led to a longer tR thanthe control polymer.

The corresponding WECs resulted to be: 60.7 � 10�3 and52.5 � 10�3 min mol L�1 g�1 for the MIP and the NIP, respectively.The equilibrium adsorption data obtained in previous paragraphsseemed to indicate that there was not an important imprinting ef-fect in the polymer synthesized against flavonol when comparedwith the control polymer. However, the observation of the break-through demonstrated clearly that the kinetics also plays animportant role in the recognition process. In fact, when using themolecular imprinted polymer against flavonol in a flow-throughapproach as optical recognition material, high fluorescence asym-metric flow-injection peaks were obtained, while symmetric peaksof low fluorescence intensity were observed when the controlpolymer was used as recognition material [14].

4. Conclusions

Elucidation of intermolecular interactions in the pre-polymeri-zation mixture is the key for the rational design of flavonol molec-ularly imprinted polymers. In this work we have demonstrated thatthe quality of the recognition binding sites in the resulting MIP is adirect function of the extent of the monomer template interactionspresent in the pre-polymerization mixture. DFT methodology atB3LYP/6-31+G�� level have applied to examine the intermolecularinteractions between flavonol and MAA. DEsolvent stabilization cal-culated by PCM predicted that chloroform is a good solvent formolecular imprinting. Synthesis was performed using different por-ogens with the aim to determine the influence of the solvent on theflavonol-monomer interactions. Results demonstrated that the im-printed polymer prepared using chloroform as porogen exhibitedspecific flavonol binding sites. The geometrical parameters involv-ing hydrogen bonding sites and the binding energies of the moststable interacting system have been determined. Results predictedthat MAA was a suitable functional monomer to generate selectiveflavonol molecularly imprinted polymers. Physico-chemical char-acterization of the MAA-based imprinted polymer (adsorption iso-therms, SEM, flow-injection analysis and adsorption dynamics)confirmed the theoretical studies.

Acknowledgments

Authors gratefully acknowledge University of Oviedo and Tech-nological Institute of Tijuana for cooperative research (2008).

M.E.D.G and J.A.G.C. acknowledge MICINN (Proj#CTQ2006-14644-C02-01/BQU). The Autonomous Metropolitan University isalso gratefully acknowledged. L. Gómez thanks CONACyT for thegranted scholarship.

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