ANALYTICAL SCIENCES AUGUST 1998, VOL. 14 699 1998 © The Japan Society for Analytical Chemistry Design and Preparation of Molecularly Imprinted Atrazine-Receptor Polymers: Investigation of Functional Monomers and Solvents Jun MATSUI, Hiroyuki KUBO and Toshifumi TAKEUCHI† Laboratory of Synthetic Biochemistry, Faculty of Information Sciences, Hiroshima City University, Ozuka-higashi, Asaminami, Hiroshima 731–3194, Japan The preparation of atrazine-receptor polymers by molecular imprinting was explored, focusing upon the role of functional monomer and solvent used for the preparation. Three functional monomers and six different solvents were examined from the viewpoint of the efficiency for molecular imprinting. Polymers were prepared using methacrylic acid, a stronger acid 2-(trifluoromethyl)acrylic acid and a three-point hydrogen bond donor/acceptor, glutethimide derivative. Methacrylic acid displayed the best effectiveness as functional monomer, which leads to an understanding that a function- al monomer is required to have both strong binding and multiple binding capacity. By choosing less polar solvents for polymerization, polymers having better retention ability and higher selectivity were obtained. Those polymers exhibited capacity factors 2 – 2.5 times as large as that for chloroform. It was also suggested that the molecular shape and size of the solvent used have influence on the resultant polymer’s binding ability. Keywords Molecular imprinting, atrazine Molecular imprinting is known as a potential atrazine-imprinted polymer receptor, focusing upon a approach to artificial antibodies/receptors and enzyme design of a functional monomer and a selection of sol- mimics. A few years ago, we reported on a molecular- vents used for molecular imprinting. ly imprinted polymer receptor for a triazine herbicide atrazine.1,2 The polymer receptor exhibited a dissocia- tion constant of 10-5 M and excellent selectivity to Experimental atrazine. Two other independent groups also reported on atrazine-imprinted polymers prepared by a similar A typical preparation of an atrazine imprinted poly- procedure3,4, where radio-ligand assay was demonstrat- mer (Fig. 1): into 25 ml of chloroform were added ed using atrazine-imprinted polymers as reagents alter- atrazine 1 (1.67 mmol), methacrylic acid 2 (6.68 native to antibodies. As another application of a mo- mmol), ethylene glycol dimethacrylate (9.35 g) and lecularly imprinted polymer, solid phase extraction 2,2¢-azobis(isobutyronitrile) (120 mg). After being using an atrazine-imprinted polymer has been reported. sonicated and sparged with nitrogen gas, the mixture The imprinted polymer has successfully demonstrated was placed under UV light at 3.5˚C for 12 h. A block its specificity for extracting a target triazine herbicide polymer obtained was crushed, sieved and packed in a from beef liver5 and from water6, showing that the tech- stainless-steel column. The polymer was washed using nique is feasible for preparing artificial antibodies/ methanol–acetic acid (7:3, v/v) to remove the template receptors usable in applications of analytical purpose. species from the polymer matrix. As functional Preparation of the atrazine-imprinted polymers1–4 was monomer, 2-(trifluoromethyl)acrylic acid 3 (6.68 carried out according to a protocol well-established by mmol) or the glutethimide derivative 4 (1.67 mmol) Mosbach, using methacrylic acid as functional was used instead of methacrylic acid. As an alternative monomer, ethylene glycol dimethacrylate as crosslink- solvent, 25 ml of toluene, o-xylene, m-xylene, p- ing agent, and chloroform or dichloromethane as sol- xylene, mesitylene or 1,4-dioxane was used instead of vent. Although the atrazine-imprinted polymers pre- chloroform, where methacrylic acid was used as the pared have shown satisfying performance up to a cer- functional monomer. Corresponding non-imprinted tain degree, further optimization of the detailed prepa- polymers were identically prepared in the absence of ration conditions is important to be conducted. Here, the template species. we report on the investigation of the preparation of Retention of atrazine was measured on the columns filled with the imprinted and non-imprinted polymers, † To whom correspondence should be addressed. using acetonitrile as the eluent at a flow rate of 1.0 ml 700 ANALYTICAL SCIENCES AUGUST 1998, VOL. 14 Fig. 1 Schematic representation of molecular imprinting of atrazine 1 using methacrylic acid 2. Structures of alternative functional monomers, 2-(trifluoromethyl)acrylic acid (TFMAA 3) and methacryloylaminoglutethimide 4, are also shown. min-1. Detection was made by UV absorbance detector at 254 nm. The capacity factor (k¢) was calculated by an equation, k¢=(tR-t0)/t0, and used for comparison of the binding ability to atrazine. Other reference sam- ples, propazine, simazine and ametryn (Fig. 2) were also examined to evaluate the selectivity. Results and Discussion Functional monomer In the molecular imprinting principle, functional monomers keep binding a template molecule during the polymerization and are complementarily arranged to the template molecule in a resultant cavity. Therefore, tailoring of a functional monomer to the template mole- cule is the most important step in molecular imprinting. Fig. 2 Structures of the triazine herbicides and other agro- In the previous study of atrazine-imprinting1,2, chemicals tested. methacrylic acid (MAA) was employed as the function- al monomer, because acetic acid has been known to form a double hydrogen bond with atrazine7 and MAA To investigate the interaction between the template was expected to be bound to atrazine in the same man- molecule, atrazine, and each functional monomer, 1H ner. Considering the acidity and multiple-interaction NMR experiments and subsequent Job’s plotting analy- ability of atrazine as important factors for an effective ses8 were performed. In 1H NMR titration of atrazine functional monomer, two functional monomers, 2-(tri- with aminoglutethimide, a down-field shift was fluoromethyl)acrylic acid (TFMAA, 3) and a observed on the sidechain amino protons of atrazine. glutethimide derivative 4, were newly tested in this Such a down-field shift was also observed on the imide study. TFMAA is a stronger acid and 4 is expected to proton of aminoglutethimide by adding atrazine into form a triple hydrogen bond with atrazine. aminoglutethimide solution, suggesting that a three- Figure 3 shows the capacity factors of atrazine point hydrogen bond was formed. However, the Job’s obtained by the three differently functionalized plot suggested that only 1:1 stoichiometry is engaged in atrazine-imprinted polymers. Unexpectedly, the the complex formation of atrazine and the monomer 4 atrazine-imprinted polymer prepared using TFMAA (Fig. 4). This could be a reason for the unsatisfactory gave a lower capacity (k¢=1.73) than the MAA-based results, because a system in which several functional atrazine imprint (k¢=3.89). Also, the glutethimide- monomers cooperatively work to imprint a template monomer 4 showed low effectiveness as a functional molecule is advantageous to develop high-affinity bind- monomer for imprinting atrazine. ing sites. The binding constant for the complexation ANALYTICAL SCIENCES AUGUST 1998, VOL. 14 701 Fig. 3 Capacity factors for atrazine in the atrazine-imprinted and the non-imprinted blank polymers. PM(At), an atrazine- Fig. 4 Job’s plot for atrazine and aminoglutethimide. A theo- imprint prepared using MAA 2; PF(At), an atrazine-imprint retical curve is drawn as K=22 M-1. prepared using TFMAA 3; PG(At), an atrazine-imprint pre- pared using the glutethimide-based monomer 4. PM(Bl), PF(Bl) and PG(Bl) are the respective non-imprinted blank polymers of the atrazine-imprints, PM(At), PF(At) and monomer to strongly bind a template molecule. PG(At). Therefore, the selection of solvents used for the polym- erization is an important factor in design of molecular imprinting system for a given target molecule. Since hydrogen bond formation is expected in the atrazine- was estimated at only 22 M-1 by 1H NMR titration, functional monomer complexation during the imprint- which would be also a cause of the poor effectiveness ing process, solvents less polar than chloroform, i.e., of 4 as the functional monomer for imprinting atrazine. toluene, o-xylene, m-xylene, p-xylene, mesitylene and In contrast, the Job’s plot obtained for MAA does not dioxane were examined. The polymers were prepared give a clear maximum when the mole fraction is 0.5 using MAA as functional monomer and tested identi- and suggests that the MAA forms several complex cally. As shown in Table 1, larger capacity factors were species with atrazine. Although Job’s plot based on 1H exhibited by the polymers prepared using less polar sol- NMR does not elucidate the stoichiometry of the com- vents as porogen. However, 1,4-dioxane showed little plexation, it is reasonable to assume that two or more capability as solvent for molecular imprinting of MAA molecules are bound to the template atrazine to atrazine, although the dielectric constant of 1,4-dioxane develop binding sites with K=10-5.2 One of the possi- is comparable to that of the other solvents tested. This ble complexes engaged in the imprinting process is weak capability could be due to 1,4-dioxane’s hydrogen shown in Fig. 1. Also, Job’s plot for TFMAA does not bond capacity interfering in complex formation of show a typical 1:1 complexation profile. Although the atrazine and MAA, which is suggested by the good sol- poor effectiveness of TFMAA for imprinting atrazine ubility of 1,4-dioxane in water unlike that of the other has not been completely understood, a plausible reason solvents. m-Xylene and mesitylene exhibited superior is the poor hydrogen bond accepting ability of TFMAA capacity factors among the benzene derivatives in spite due to the electron attracting trifluoromethyl group, of their similar dielectric constants. It is also notable which would make it less possible for TFMAA to form that high atrazine-selectivity was demonstrated by these a double hydrogen bond favorable in terms of entropy.