Anal. Chem. 2000, 72, 1810-1813
Atrazine-Selective Polymers Prepared by Molecular Imprinting of Trialkylmelamines as Dummy Template Species of Atrazine Jun Matsui, Kuniyuki Fujiwara, and Toshifumi Takeuchi*
Laboratory of Synthetic Biochemistry, Faculty of Information Sciences, Hiroshima City University, 3-4-1 Ozuka-higashi, Asaminami-ku, Hiroshima 731-3194, Japan
Synthetic polymer receptors selective for atrazine have been prepared by molecular imprinting using trialkylmelamines as template molecules in place of atrazine. Trialkylmelamines were shown to be useful as templates for introducing affinity for atrazine into ethylene glycol dimethacrylate-methacrylic acid copolymers. The polymers showed the selective binding capacity for triazine herbicides including atrazine, whereas agrochemicals in other categories were not adsorbed to the imprinted polymers. The group selectivity demonstrated was comparable with that of the original atrazine imprinted polymers. Use of the nonagrochemical template molecules as a substitution to atrazine has made it possible to synthesize herbicide-receptor polymers free from troubles caused by analyte contaminants, which are desired for analytical applications. Molecularly imprinted polymers selective for atrazine have been recognized as artificial antibodies/receptors potentially useful for analytical applications. Recent papers, reporting on the synthesis,1-4 mechanism,5 assays,2,3 sensors,6,7 and solid-phase extraction (SPE),8-10 have demonstrated the high affinity and specificity of the imprinted polymers available in organic solvents and promised the usefulness of the imprinted polymers for various analytical applications. Principally, molecular imprinting tailors polymers to a template molecule added in a polymerization mixture. To date, therefore, atrazine-selective polymers have been synthesized by molecular imprinting using atrazine itself as the * To whom correspondence should be addressed. Phone: +81-82-830-1603. Fax: +81-82-830-1610. E-mail:
[email protected]. (1) Matsui, J.; Miyoshi, Y.; Doblhoff-Dier, O.; Takeuchi, T. Anal. Chem. 1995, 67, 4404-4408. (2) Muldoon, M. T.; Stanker, L. H. J. Agric. Food Chem. 1995, 43, 1424-1427. (3) Siemann, M.; Andersson, L. I.; Mosbach, K. J. Agric. Food Chem. 1996, 44, 141-145. (4) Matsui, J.; Kubo, H.; Takeuchi, T. Anal. Sci. 1998, 14, 699-702. (5) Dauwe, C.; Sellergen, B. J. Chromatogr. 1996, 753, 191-200. (6) Piletsky, S. A.; Piletskaya, E. V.; Elgersma, A. V.; Yano, K.; Karube, I.; Parhometz, Y. P. Biosens. Bioelectron. 1995, 10, 959-964. (7) Sergeyeva, T. A.; Piletsky, S. A.; Brovko, A. A.; Slinchenko, E. A.; Sergeeva, L. M.; Panasyuk, T. L.; Elskaya, A. V. Analyst (Cambridge, U.K.) 1999, 124, 331-334. (8) Muldoon, M. T.; Stanker, L. H. Anal. Chem. 1997, 69, 803-808. (9) Matsui, J.; Okada, M.; Tsuruoka, M.; Takeuchi, T. Anal. Commun. 1997, 34, 85-87. (10) Bjarnason, B.; Chimuka, L.; Ramstro¨m, O. Anal. Chem. 1999, 71, 21522156.
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template species. It is a great concern, however, in analytical applications that atrazine used as the template may remain in the washed polymers and seep out of the polymers in use. In actuality, it appeared that the removal of the template atrazine is extremely time-consuming and cannot be practically completed in our preliminary studies.11 Contamination of analytical samples by atrazine fatally interferes with precise quantitation and blinds sensitive detection of atrazine. Thus, the synthetic protocol using the analyte itself leaves serious questions about the reliability of the analytical protocol using molecularly imprinted polymers, providing a barrier for imprinted polymer-based SPE to become an established method. Therefore, molecular imprinting using template species substitutive to atrazine is desired for developing atrazine-selective polymers truly useful for practical environmental analysis. In the previous synthesis of atrazine-imprinted polymers, atrazine was expected to form hydrogen bonds with methacrylic acid (MAA) used as the functional monomer. Therefore, here we propose trialkylmelamines as the template species substitutive to atrazine, bearing functional groups capable of hydrogen bonding with MAA in a similar fashion. In this study, we report on molecularly imprinted polymers prepared using triethylmelamine (TEM), triisopropylmelamine (TPM), and tributylmelamine (TBM) as dummy template species substitutive to atrazine. EXPERIMENTAL SECTION Materials and Equipment. Triethylmelamine (TEM), tributylmelamine (TBM), triisopropylmelamine (TPM), and atrazine were kindly donated by Nissan Chemical Industries (Tokyo, Japan). Methacrylic acid (MAA), ethylene glycol dimethacrylate (EDMA), 2,2′-azobis(isobutyronitrile) (AIBN), and other triazine herbicides were obtained from Wako Pure Chemical Industries (Osaka, Japan). Chloroform, acetonitrile, methanol, and acetic acid were purchased from Katayama Chemical Industries (Osaka, Japan). Before use, MAA, EDMA, and chloroform were purified by standard procedures.12 Empty columns were purchased from GL Sciences (Tokyo, Japan). A UV lamp (UVP, Upland, CA, XX-15L) and a cooling circular bath (EYELA, Tokyo, CA-111) were used for synthesizing the polymers. A mechanical mortar (Nitto Kagaku, Tokyo, ANM-1000) (11) Unpublished results. (12) Perrin, D. D.; Armarego, W. L. F. Purification of Laboratory Chemicals, 3rd ed.; Pergamon Press: Oxford, 1988. 10.1021/ac9911950 CCC: $19.00
© 2000 American Chemical Society Published on Web 03/17/2000
and stainless sieves (Iida Manufacturing, Osaka, Japan) were used for grinding and sieving polymers, respectively. Liquid-chromatographic analyses were performed using a Waters Millennium system (Milford, MA) consisting of a pump (600s), a photodiodearray detector (996), and an automatic sample injector (717 plus). Preparation of Imprinted Polymers. A typical preparation for the triethylmelamine-imprinted polymer P(TEM): into chloroform (25 mL) were added TEM (351 mg, 1.67 mmol), MAA (567 µL), EDMA (8.89 mL), and AIBN (120 mg). The mixture in a glass tube was sonicated and bubbled with nitrogen gas, before being sealed, and placed under UV light at 5 °C for 12 h. The block polymer obtained was crushed and ground in a mortar. Other imprinted polymers, P(TPM) and P(TBM), were prepared identically using TPM and TBM instead of TEM, respectively. A nonimprint blank polymer, P(blank), was prepared in the absence of template species. Polymer Evaluation by Chromatography. Polymer particles were suspended in acetonitrile-chloroform (1:1, v/v) and packed in a stainless steel column (100 mm × 4.6 mm, i.d.) using a chromatographic pump at a flow rate of 9 mL min-1. The column was washed with 300 mL of methanol-acetic acid (7:3, v/v) at a flow rate of 0.5-1.0 mL min-1. Retention time was recorded, after the column was conditioned with the eluent, by independent injections of the template compound, atrazine and other triazine herbicides, and structurally nonrelated agrochemicals. Sample size and concentration were, respectively, 20 µL and 1.0 mM. Elution was monitored by UV absorbance using a photodiode-array detector (230-270 nm, max). A retention factor (k) was obtained by an equation k ) rs/(rs - ra), where rs and ra, respectively, represent a retention time of a sample and that of acetone as a void marker. RESULTS & DISCUSSION Alkyl-Substituted Melamines as Dummy Template Species. Atrazine as a template molecule in methacrylic acid (MAA) based molecular imprinting has been studied by several groups.1-4 Dual hydrogen bonds are expected to be formed between atrazine and MAA as a key interaction necessary for binding-site construction, whereby a carboxylic group of MAA works as both a hydrogen-bond acceptor and a donor interacting with a hydrogen atom of the amino group and a nitrogen atom of the triazine body, respectively (Figure 1A).13 Although no direct evidence has been shown, it would be reasonable to assume that two or more hydrogen bonds are formed with MAA and maybe with EDMA molecules during the imprinting process, judging from the highly selective binding property of the resultant imprinted polymers.1-3 Therefore, templates substitutive to atrazine should possess two or more dual-hydrogen bondable functional groups. The chloro substituent of atrazine is known to play important roles in expressing the biological activity. Because dummy template molecules are preferred to be nonagrochemicals, in respect to the use of the resultant polymers for assaying agrochemicals, the chloro substituent should be replaced by another functional group. The substituents at the position also have crucial influences, possibly due to electron-withdrawing or donating effects, on the basicity of the nitrogen atoms and the consequent fashion of complexation with functional monomers.5 Thereby, the (13) Welhouse, G. J.; Bleam, W. F. Environ. Sci. Technol. 1993, 27, 494-500.
Figure 1. Schematic illustration of possible intermolecular interactions with methacrylic acid (MAA) and ethylene glycol dimethacrylate (EDMA): 1, atrazine; 2, triethylmelamine (TEM). R is -C(CH3)dCH2 and R′ is -C2H4-OCO-C(CH3)dCH2.
previously reported atrazine-imprinted polymers showed the selectivity toward triazine herbicides bearing the same chloro substituent, e.g., simazine and propazine, rather than those having a methylthio or a methoxy group instead, e.g., ametryn and atraton. In this sense, dummy template species are required to be similar in basicity to atrazine bearing a chloro group. Considering these factors, triethylmelamine (TEM), triisopropylmelamine (TPM), and tributylmelamine (TBM) were examined as the template species substitutive to atrazine (Table 1). These melamine-based dummy template molecules bear two alkylamino groups mimicking triazine herbicides and the third amino group instead of the chloro group, potentializing the interaction with three MAA/EDMA molecules through hydrogen bonds (Figure 1B) and allowing no biological activities comparable with that of atrazine. Fortunately, the amino groups are conceived to show similar electron effects to the chloro group, i.e., inductive electron withdrawal and conjugative electron donation. Therefore, it can be expected that the dummy molecules form atrazine-like complexes with MAA. Synthesis of Dummy Molecular Imprints. Molecular imprints were prepared using TEM, TPM, and TBM as template species. The recipe was adopted, following our previous one for preparing atrazine-imprinted polymers,1 whereby four molar equivalents of MAA to the template are used as the functional monomer. Retentions of the alkylmelamine species were compared in liquid chromatography using the imprinted and the blank polymers as stationary phases to ensure that the template species were satisfactorily imprinted. The eluent used for the assessment was carefully chosen to elute all the alkylmelamines in an appropriate retention time. Chloroform, used for synthesizing the polymer, would be the most favorable solvent for the rebinding of the template to the binding sites; however, no clear elution was observed using the eluent. Therefore, we tested a more polar Analytical Chemistry, Vol. 72, No. 8, April 15, 2000
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Table 1. Structures of Triazine Herbicides and Dummy Template Molecules
Table 3. Retention (k) of Triazine Herbicides by Dummy Molecular Imprints polymer
compound
category
R1
R2
X or R3
atrazine simazine propazine cyanazine terbutylazine ametryn prometryn terbutryn triethymelamine (TEM) tributylmelamine (TBM) triisopropylmelamine (TPM)
A A A A A A A A B
Et Et i-Pr Et Et Et i-Pr Et Et
i-Pr Et i-Pr C(CH3)2CN t-Bu i-Pr i-Pr t-Bu Et
Cl Cl Cl Cl Cl SCH3 SCH3 SCH3 Et
B
n-Bu
n-Bu
n-Bu
B
i-Pr
i-Pr
i-Pr
Table 2. Retention (k) of the Original Templates by Dummy Molecular Imprintsa polymer sample
P(TEM)
P(TBM)
P(TPM)
P(blank)
TEM TBM TPM
8.5 7.9 2.2
5.2 6.1 3.2
3.2 3.8 4.2
0.54 0.58 0.13
a
Acetonitrile-acetic acid (94:6, v/v) was used as the eluent.
solvent, acetonitrile, for moderately breaking the hydrogen bonding between the substrate and the binding sites. Acetic acid was used along with acetonitrile to tune the retention time by competing against the carboxylic residues for binding with the substrate. With an increase of acetic acid, retention of the template species was decreased (no data shown), supporting the fact that the retention observed is predominantly based upon hydrogen bonding or electrostatic interaction with carboxylic residues in the polymer. The retention factors obtained using acetonitrileacetic acid (94:6, v/v) were listed in Table 2. All the imprinted polymers exhibited longer retention for all the alkylmelamines tested compared with that of the nonimprint polymer P(blank), showing that the template molecules were effective for enhancing retention ability of the polymers. Furthermore, each molecularly imprinted polymer exhibited the largest retention factor for the original template species. The selectivity observed suggests that the template molecules were certainly imprinted. Among the three imprinted polymers, P(TEM) showed the largest retention factor for the corresponding template compound, whereas P(TPM) gave the shortest retention of TPM. Difference of the template molecules in the effectiveness for developing the retention ability and selectivity can be explained by length and bulkiness of the side chains. The shortest alkyl chains of TEM could be favorable for the interaction of the template molecule with MAA during the polymerization. Accord1812 Analytical Chemistry, Vol. 72, No. 8, April 15, 2000
samplea
P(TEM)
P(TBM)
P(TPM)
P(blank)
atrazine simazine propazine cyanazine ametryn prometryn terbutylazine terbutryn 1,3,5-triazine atrazineb
10.4 11.5 9.0 6.7 9.9 6.9 8.4 5.2 0.29 0.46
8.2 8.8 7.3 5.3 6.6 5.2 7.4 3.8 0.10 0.31
6.3 6.4 8.6 5.3 9.0 7.5 6.9 3.9 0.11 0.34
0.66 0.75 0.61 0.62 0.88 0.77 0.39 0.50 0.08 0.25
a Chloroform-acetonitrile (3:1, v/v) was used as the eluent. b Acetonitrile-acetic acid (94:6, v/v) was used as the eluent.
ing to the results, the isopropyl groups of TPM would be more obstructive for the complex formation than the longer butyl groups of TBM. Atrazine Selectivity of Dummy Molecular Imprints. Retention of atrazine and other triazine herbicides by the melaminederivative-imprinted polymers was investigated by liquid chromatography. All the imprinted polymers retained the triazine herbicides tested, while no significant retention was observed in P(blank) as seen in Table 3. Among the three imprinted polymers, P(TEM) exhibited the largest retention factor for atrazine. Also, P(TEM) showed larger retention factors than P(TPM) and P(TBM) did for most of the triazine herbicides tested. The higher retention ability of P(TEM), compared with those of P(TPM) and P(TBM), could be explained by the fact that the shorter side chains of TEM only mildly disturb the template-functional monomer complexation, a fact which is supported by the superior template retention ability of P(TEM). In contrast, the relatively long n-butyl groups of TBM would be unfavorable for the complexation and the bulkiness of the isopropyl groups may hinder the neighbor amino group more critically from forming hydrogen bonds with MAA in the pre-polymerization mixture, leading to the poor retention ability for atrazine. In all the imprinted polymers, the retention atrazine displayed was lower than that of the original template species. These results are reasonable because the binding sites are conceived to be tailored to the original templates according to the molecular imprinting principle. The results would also be accounted for by the different number of potential interaction points between the triazine herbicides and the melamine derivatives. The affinity of the triazine herbicides could be inherently lower due to the deficient number of amino groups, which is suggested by the shorter retention of the triazine herbicides in P(blank) compared with those of the alkylmelamines. It is interesting to note that no distinct selectivity was observed for the triazine herbicides with a chloro group (Cl-triazine) or those with a methylthio group (SCH3-triazine); Cl triazines were retained longer in P(TEM), similarly in P(TBM), and shorter in P(TPM), compared with SCH3 triazines. It has been known that the substituent, Cl or SCH3, of the triazine-herbicide template molecule has an influence on the selectivity of the resultant polymers; for instance, ametryn-imprinted polymers are selective for prometryn having the same SCH3 substituent rather than atrazine bearing the same alkyl side chains. In the preparation of
Table 4. Retention (k) of Structurally Unrelated Agrochemicalsa polymer sample
P(TEM)
P (TBM)
P(TPM)
P(blank)
atrazine asulam mecoprop propyzamide iprodione thiram alachlor bentazone MCP ethyl metribuzin pendimethaline MBPMC TPN diuron diadinon flutanil
10.4 0.2 0.2 0.0 0.0 0.0 0.0 0.2 0.0 0.1 0.0 0.0 0.0 0.2 0.0 0.1
8.2 0.3 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
6.3 0.2 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0
0.66 0.2 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
a
Chloroform-acetonitrile (3:1, v/v) was used as the eluent.
these dummy imprints, the alkylamino group was engaged in the imprinting process instead of Cl or the SCH3 group. It would be reasonable to assume that the retention characteristic of each polymer arose from the bulkiness of the alkylamino group rather than the electron property, because the bulkiest TPM gave the best selectivity for SCH3 triazinesslarger than Cl triazines. The results also demonstrated that the dummy molecular imprinting is useful for developing selectivity for a group of chemical species while the original molecular imprinting is suitable for obtaining specificity to the exact template compound. No clear effects of the alkyl groups of the template species on the resultant size/ shape recognition of side chains of the triazine herbicides were observed. Simazine was retained significantly longer than propazine by P(TEM) and the opposite results were obtained in P(TPM), suggesting that the molecular imprinting can develop recognition of the side chains of triazine herbicides. P(TBM) bearing n-butyl groups, however, showed the longest retention for the smallest simazine. Also, P(TPM) prefers ametryn rather than prometryn. Thus, the origin of the recognition property brought about by dummy species imprinting has been currently understood only partially.
Selectivity Against Agrochemicals in Other Categories. The most important feature of imprinted polymers in terms of analytical applications is selectivity for an analyte against contaminants in samples rather than absolute binding strength for an analyte. Therefore, the selectivity of the alkylmelamineimprinted polymers was further investigated to assess the practical availability of the imprinted polymers for analytical use. Retention time of nontriazine agrochemicals was measured on P(TEM), P(TPM), and P(TBM). As shown in Table 4, no samples exhibited significant retention, displaying instead the highly selective retention characteristics of the imprinted polymers for the triazine family. Although the retention factor of atrazine in the dummy atrazine receptor is currently lower than that in the genuine atrazine-imprinted polymer examined in our previous study,1 the group selectivity obtained here is comparable, encouraging the application of these dummy molecular imprints for practical analyses of triazine herbicides. CONCLUSIONS Molecular imprinting using dummy template species has been shown to be available for developing polymers selective for triazine herbicides. Dummy molecular imprinting will increase the availability of the molecular imprinting technique in a variety of application fields, especially when the presence of residual template species causes serious concerns and the analytes are inappropriate compounds for massive use as a template because of toxicity or costs. Also, systematic studies on the relation between template species and resultant selectivity will be helpful for understanding the molecular imprinting and recognition mechanism. In our laboratory, further studies seeking more effective dummy template species and applying the dummy atrazine-selective polymers are ongoing. ACKNOWLEDGMENT We thank Dr. Kenji Makino (Nissan Chemical Industries) for many helpful discussions. The financial support from The Ministry of Education, Science, Sports and Culture of Japan is gratefully acknowledged. This work is also funded by Hiroshima City University Grant for Special Academic Research. Received for review October 18, 1999. Accepted January 18, 2000. AC9911950
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