Development of molecularly imprinted polymer membranes with specificity to triazine herbicides , prepared by the « surface photografting » technique

«Surface photografting» of polypropylene (PPy) microporous membranes by molecularly imprinted polymers selective to triazine herbicides has been carried out by the UV irradiation-initiated copolymerization of the functional monomer (2-acrylamido-2-methyl-l-propane sulphonic acid) and a cross-linker (N,N'-methylene-bis-acrylamide) in the presence of the template (terbumeton) onto photoinitiator (benzophenone)-coated samples. The grafting reaction occurs in a thin liquid layer on the membrane substrate, which is pre-soaked in a dimethyl formamide solution containing template, functional monomer and cross-linker. After irradiation with a 500 W mercury lamp for 10 min at room temperature, the membranes covered with the layer of imprinted polymer were obtained. The recognition sites complementary to terbumeton were formed in the membranes after extraction of the template molecules with methanol. Alternatively, reference polymeric membranes were prepared with the same monomer composition, but without the template. The membranes' recognition properties were estimated by their capability to herbicide adsorption from its aqueous solution. The membranes modified by the mixture of monomers containing terbumeton showed significantly higher adsorption capability to this herbicide than to analogous compounds (terbuthylazine, atrazine, desmetryn, metribuzine). The effect of the polymer composition on the binding properties of the membranes has been investigated. High affinity of these membranes to triazine herbicides together with their inexpensive preparation, provide a good basis for applications of molecularly imprinted polymer membranes in separation and solid-phase extraction


«Surface photografting» of polypropylene (PPy) microporous membranes by molecularly imprinted polymers selective to triazine herbicides has been carried out by the UV irradiation-initiated copolymerization of the functional monomer (2-acrylamido-2-methyl-l-propane sulphonic acid) and a cross-linker (N,N'-methylene-bis-acrylamide) in the presence of the template (terbumeton) onto photoinitiator (benzophenone)-coated samples. The grafting reaction occurs in a thin liquid layer on the membrane substrate, which is pre-soaked in a dimethyl formamide solution containing template, functional monomer and cross-linker. After irradiation with a 500 W mercury lamp for 10 min at room
temperature, the membranes covered with the layer of imprinted polymer were obtained.

The recognition sites complementary to terbumeton were formed in the membranes after extraction of the template molecules with methanol. Alternatively, reference polymeric membranes were prepared with the same monomer composition, but without the template. The membranes' recognition properties were estimated by their capability to herbicide adsorption from its aqueous solution. The membranes modified by the mixture of monomers containing terbumeton showed significantly higher adsorption capability to this herbicide than to analogous compounds (terbuthylazine, atrazine, desmetryn, metribuzine). The effect of the polymer composition on the binding properties of the membranes has been investigated. High affinity of these membranes to triazine herbicides together with their inexpensive preparation, provide a good basis for applications of molecularly imprinted polymer membranes in separation and solid-phase extraction
Introduction.In many applications of bioorganic and environmental chemistry it is desirable to utilize a membrane filter, which is mechanically strong, ther mally stable, insoluble in most organic solvents, relatively inert chemically and has affinity to certain analyte [1][2][3].The binding properties of affinity membranes are determined by specific receptor-ligand interactions.However, synthesis of the membranes selective for a broad range of neutral molecules is challenged by the difficulty of incorporating specific selectivity to low-molecular weight compounds [7][8], very good thermal and mechanical stability [9 ], their production is inexpensive.Polymers of this type have been used as sta tionary-phase materials to make highly selective liq uid chromatography columns [10], as substitutes for antibodies in competitive binding assays [11][12][13], and as selective elements of chemical sensors [14].Chromatographic and SPE applications traditionally utilize МІР particles prepared by grinding and sieving of synthesized polymer blocks or the particles pre pared by suspension polymerization.The first ap proach is time consuming, may lead to the destruction of some binding sites in the polymer and produces a relatively low yield of the fraction with a narrow size distribution.In the second approach, the choice of monomers is limited to those, which are not soluble in the dispersion phase.Additionally the synthesized beads are not always uniform in their shape and size.Thus, again a sieving procedure is required which makes column packing time-consuming, expensive and ineffective.Use of polymer particles of a small size in chromatography is also associated with too high backpressure.Therefore, the method of mo lecular imprinting was also combined with the mem brane technology in order to develop new generation of stable affinity membranes for the separation of the target molecules from a mixture of structurally similar compounds [15][16][17].However, the high selectivity and stability of these membranes were shaded by their ineffective performance, particularly due to small fluxes.Typically, the fluxes observed were not larger than 10~4 mol-m" 2 -h _1 [18].This effect can be related to the high degrees of cross-linking of MIPs, which is a prerequisite for the imprinted membranes selectivity.
Other approaches to the synthesis of affinity membranes are chemical or the photografting of a thin layer of the imprinted polymer to the surface of a porous membrane.There have been two recent re ports on the polymers [19] and membranes [20] grafted with MIPs.In the last case the laboratorymade membranes from photo-reactive polymer (polyacrylonitrile-co-diethylaminodithiocarbamoyl methylsty rene) were grafted with acrylic acid and N,N'methylene-bis-acrylamide in the presence of theo phylline yielding theophylline-specific membranes.Unfortunately, the use of a special polymer for the membranes formation together with long reaction times for МІР functionalization (24 h) substantially reduce the areas of the membranes' applications.Much more efficient method for the membrane surface functionalization is currently being explored [21 ].
The aim of the present research is the develop ment of a general method for molecular imprinting the surface of a stable synthetic polymer membrane.Surface photograft co-polymerization in the presence of a template should introduce specific binding sites into the porous membrane without damaging its pore structure and, thus, preserving its transport pro perties.
The present paper describes the procedure of modification of commercially available microporous membranes with a herbicide-imprinted polymer using technique of «surface photografting» from organic solvents.This substantially broadens the number of potential templates allowing one to use a wide range of substances poorly soluble in aqueous environment.The membranes modified with the imprinted poly mers possess high selectivity to the analyte without significant change in their original permeability.
Preparation of membranes modified by molecularly imprinted polymers.Circular PPy or PA mem brane samples (A = 5 cm 2 ) were extracted with chlo roform in Soxhlet apparatus during 4 h, dried and weighted.The membranes were then pre-soaked in 0.15 M solution of BP in acetone for 5 min and dried under vacuum.The pre-coated with photoinitiator membranes were transferred to a dimethyl formamide (DMF) solution, containing 10 mM of terbumeton, 50 mM of functional monomer (acrylic, methacrylic or 2-acrylamido-2-methy 1-1 -propane sulphonic acid), 150-500 mM of N,N'-methylene-bis-acrylamide.To prevent desorption of the photoinitiator from pre viously coated membranes 5 mM of BP was added to the monomer mixture.The pre-soaked for 5 min in the monomer mixture membranes were then UV irradiated on a pilot-scale UV curing system (500 W mercury lamp; Beltron GmbH, Germany) for 10 cycles (1 cycle = 1 min).To remove the homopolymer, residual chemicals and template, the resulting mem branes were extracted by hot methanol in Soxhlet apparatus for 2 hours.After drying the membranes were weighted again and the degree of modification (DM) was calculated from weight differences.

Membranes' characterization.
The binding pro perties of the membranes were estimated by their capability to herbicide adsorption from its aqeous solution.Adsorption of the herbicide from water by the membrane was estimated in filtration experiments using syringe connected to a filtration cell holder (d = = 25 mm, «Schleicher & Schulb, Germany).In the adsorption experiments 10 ml of 5 10" 7 -10~4 M herbicide solution were filtered through the mem branes, typically at a rate of 10 ml/min.The filtrate was extracted with 10 ml of chloroform.The herbicide concentrations in both feed and permeate solutions were determined by gas chromatography after the extraction procedure using Hewlet Packard GC sys tem HP 6890 with the mass selective detector HP 5973 (column HP5MS).
Results and Discussion.During the last decades, within the field of surface modification of various substrates, photografting from organic solvents has received wide attention [22][23][24][25].In general, such technique has involved immersion of the substrate to be grafted in a solution of monomer in an organic solvent, and subsequent exposure to irradiation.Such photo-induced grafting is accompanied by extensive homopolymerization and often causes uneven modi fication of the substrate surface.Recently Ranby et al. have proposed a new method of «surface photo-

The scheme of the «surphotografting» modificatiof the porous membrane molecularly imprinting po-
grafting» for polymer modification, where the sub strate is pre-soaked in a solution of monomer and then UV-irradiated in an inert atmosphere [26].According to this technique, the grafting reaction occurs in a thin layer of the solution on the substrate surface.Little homopolymerization occurs under these grafting conditions and small amounts of the formed homopolymer can be removed by the washing proper procedure.
To modify PPy and PA membranes by a thin layer of the imprinted polymer, the method of «surface photografting» has been applied.The main reason for this is very high reactivity of the crosslinker (N,N'-methylene-bis-acrylamide) and a prob lem of its homopolymerization in heterogeneous reac tion systems.This generally leads to low degrees of the membranes' modification, poor reproducibility and difficulty in recovering copolymer in the noncontaminated form, especially at high grafting yields.The scheme of the «surface photografting» modifi cation of the porous membrane with МІР is presented in the Fig. 1.In this polymerization scheme, the stabilizing effect of highly cross-linked molecularly imprinted polymer in a combination with a flexible, chemically inert and wide pore PPy or PA membrane is utilized.
It is reasonable to assume, that the composition of the monomer mixture determines the ability of the resulting membranes to bind the template selectively and, thus -the membranes adsorption capacity.Therefore, the influence of the type and concentration of the functional monomer and cross-linker, as well as the template concentration in the monomer mixture, on the ability of the modified membranes to effective adsorption of the template has been investigated.

Influence of a type of the functional monomer on the membranes adsorption capability.
Modification of the PPy membranes with imprinted polymers was performed using acrylic, methacrylic or 2-acrylamido-2-methyl-l -propane sulphonic acids as functional mo nomers and N,N'-methylene-bis-acrylamide as a cross-linker.Triazine herbicide terbumeton was used as a model template in the present research.A set of the imprinted and reference membranes with AA, MA or AMPS as functional monomers has been obtained and tested in filtration experiments.AA and MA were demonstrated to be ineffective as functional mono mers.The imprinted membranes prepared in the presence of AA and MA as functional monomers demonstrated either the same or lower adsorption capability as reference ones.The membranes imp rinted with terbumeton and prepared in the presence of AMPS as a functional monomer demonstrated significantly higher adsorption capability than the reference membranes of the same composition (Fig. 2).It is assumed that AMPS as a strong acid (pKa< < 1) can protonate terbumeton (pKa = 4.2) and an ion-pair complex is formed between the template and AMPS in the initial monomer mixture.For weaker acidic monomers MAA (pKa = 4.65) and AA (pKa = = 4.2) this mechanism is less effective.This has been also confirmed by the investigations of the template-functional monomer complex formation by UV-difference spectroscopy.These data verify that the complex AMPSA-terbumeton is significantly stronger (K dis = 3.0 10~5 ± 0.3 10" 5 M) as compared to AA (K dis =2.010" 4 ± 0.3-10" 4 M) and MAA (K dis = = 8.010" 5 ±1.010" 5 M).
Influence of the cross-linker concentration on photografting and the membranes' adsorption capa bility.As widely recognized, the effective performance of the imprinted polymer is provided by high degrees of the polymer cross-linking.In this case, the selective cavities can retain their shapes even after extraction of the template.At the same time, a certain degree of the polymer chains' flexibility is important to provide rapid equilibration with the template to be bind.Hence, the influence of a type and amount of the monomer and cross-linker in the monomer mixture used for the membranes modification on both the degree of modification and adsorption capability of the resulting membranes was investigated.The former was explored as a function of the cross-linker con centration (N,N'-methylene-bis-acrylamide) in the monomer mixture.
The degree of the modification was shown to increase with the increase in the cross-linker con centration in the monomer mixture and to comprise 150-500/^g/cm 2 of membrane surface (Fig. 3, a).At the same time, the increase in MBAA concentration up to 225 mM caused also the increase in the membranes' adsorption capability (Fig. 3, b).How ever, further increase in the MBAA caused a decrease in the membranes' adsorption capability.To increase the specific adsorption of the herbicide by МІР § 500  membranes, i. e. to decrease the level of nonspecific binding, the other cross-linker (trimethylolpropan trimethacrylate) was used.Although the degrees of modification were higher, than in the case of N,N'methyiene-bis-acrylamide, the values of specific ad sorption were twice lower.This indicates, that high degrees of modification together with excessive deg rees of cross-linking are not necessary for the ef fective performance of МІР membranes.Evidently, at these conditions most of the template molecules are trapped in the excessively cross-linked domains of imprinted polymer and not accessible for the reac tions, which results in a decreased amount of the binding sites.Structure of the binding sites.The AMPS con centration in the monomer mixture was varied to optimize the ratio between the template and a func tional monomer used for the membranes' modifi cation.No difference between adsorption capability of imprinted and reference membranes was obtained in the absence of AMPS in the monomer mixture or when its concentration was too low (10 mM).Ob->etween terbumeton adsorption on а МІР membrane and a reference )f the template.The «zero» values of specific adsorption correspond reference membranes or to the cases of preferential adsorption of viously, these ratios yield the imprinted polymers with insufficient extents of the template complexation and, thus, low number of binding sites.Use of AMPS in concentrations of 10-50 mM produced МІР mem branes with the improved adsorption capability as compared to reference ones.However, the further increase in AMPS concentration (60-80 mM) leads to formation of the membranes with too high degrees of non-specific binding (Fig. 4).Apparently, this can be explained by an abundance of the polar functional groups distributed randomly throughout the polymer matrix, that results in the reduced selectivity.

Influence of the template concentration.
To define the optimal number of binding sites in the resulting membranes, influence of the template concentration on the membrane adsorption capability has been studied.The highest value of specific adsorption was observed at the template concentration of 10 mM.Both decrease and increase in the template concen tration resulted in either lower or no imprinting effects (Fig. 5).Evidently, a decrease in the template concentration results in the insufficient number of the No imprinting effect at 20 mM of terbumeton in the monomer mixture can be explained by its ability to form aggregates in the solution of high concentrations, that results in for mation of the increased number of non-and weaklyselective binding sites.
Influence of the support.Importantly, PA mem branes modified by the molecularly imprinted poly mer layer under optimized conditions, demonstrated higher degree of modification as compared to PPy membranes.However, no improvement in the her bicide binding was achieved.Practically no difference or even preferential herbicide adsorption was ob served for reference membrane as compared to the imprinted one (Table ).This effect can be related to the increased ability of PA membranes to swell in organic solvents in contrast to PPy membranes.Since there is no swelling of PPy membranes in organic solvents, we can assume that the functionalization takes place in a thin layer on the entire surface of the membrane.In contrast to this, in the case of PA membranes the polymerization reaction takes place in the entire membrane volume, i. e. mainly in the swollen PA matrix.Therefore, the degrees of mo dification achieved under same conditions are sig nificantly higher for PA than for PPy (Table ).However, one can assume that most of the polymer entrapped in PA is not accessible for binding with the template molecules during the fast filtration step.From the other side, swelling of the support changes the three-dimensional configuration of the functional groups participating in the recognition process and may lead to the loss of selectivity.The other exp lanation can be interaction between amide func tionalities in the PA support and the functional monomer, that hinders binding the herbicide by the functional monomer.

Specificity of molecularly imprinted polymer mem branes and their herbicide-binding efficiency.
A series of terbumeton analogs were used to examine the selectivity of the obtained membranes.The capability of the terbumeton-imprinted membranes to bind her bicides of the related chemical structure was tested in filtration experiments.The terbumeton-imprinted membranes were shown to be capable of binding terbumeton analogs much less effectively than ter bumeton (Fig. 6).It was demonstrated that the terbumeton-imprinted membranes modified under the optimized conditions were able to recover 95-99 % of terbumeton from its 5-Ю" 7 -10~4 M aqueous so lutions (Fig. 7).The adsorption capability of the terbumeton-imprinted membranes was determined in filtration experiments using saturation with 10~5 M solution of terbumeton.The value determined was 5 jug/cm 2 (-22 nM/cm 2 ), which corresponds to ap proximately 40 % of the theoretical value calculated from the degree of modification and the stochiometry of the template-functional monomer complex.The refore, they can be successfully used for both water purification and herbicide pre-concentration in en vironmental analysis.
Conclusions.The new type of composite membra nes, having artificial recognition sites for terbumeton, was prepared by «surface photografting» of 2-acrylamido-2-methyl-l-propane sulphonic acid, N,N'methylene-bis-acrylamide in the presence of ter bumeton as a template on a benzophenon-coated polypropylene 0.2 jum membranes.The membranes imprinted with terbumeton demonstrated significantly higher adsorption capability to this herbicide than to analogous compounds (terbuthylazine, atrazine, des metryn, and metribuzine).No affinity for terbumeton was observed for МІР-photografted polyamide mem branes, which indicates significant influence of the support on both the imprinting procedure and the process of template recognition.The type and con-  centration of the functional monomer as well as the concentration of cross-linker have a crucial influence on the resulting membranes' adsorption capability.High affinity of these membranes to triazine her bicides together with their simple and inexpensive preparation, provides a good basis for the applications of imprinted polymers in separation, solid-phase ex traction, and in a pre-concentration step for the determination of photosynthesis-inhibiting herbicides in water.Acknowledgements.Financial support from AIF, INTAS (grant YSF-00-25), and National Academy of Sciences of Ukraine is gratefully acknowledged.

Fig. 2 .
Fig. 2. Influence of the type of the functional monomer on the specific adsorption of terbumeton on the imp rinted membranes, 10 ml of 10~5 M aqueous solution of terbumeton was used in filtration experiments (see note to the Table)

Fig. 3 .Fig. 4 .Fig. 5 .
Fig. 3. Dependence of the degrees of modification of the imprinted (gray bars) and reference (white bars) membranes on the concentration of the cross-iinker in the monomer mixture (a).Dependence of the specific adsorption of terbumeton on the imprinted membranes on the concentration of the cross-linker in the monomer mix ture (b) (see note to the Table).Templa te -terbumeton, 10 mM, functional mo nomer -2-acrylamido-2-methyl-l-pro pane sulphonic acid, 50 mM, cross-lin ker -N,N'-methylene-bis-acrylamide, 150-400 mM value of specific adsorption corresponds to the difference t one, prepared with the same monomer composition but in the absence с either to the cases of the same adsorption of terbumeton on МІР and terbumeton on reference membranes.