Anal. Chem. 1998, 70, 943-945
Molecularly Imprinted Chiral Stationary Phase Prepared with Racemic Template Ken Hosoya,* Yuichi Shirasu, Kazuhiro Kimata, and Nobuo Tanaka
Department of Polymer Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan
The first example of molecularly imprinted chiral stationary phase prepared using a racemic template is shown. N-(3,5-Dinitrobenzoyl)-r-methylbenzylamine (DNB) was chirally discriminated on the molecularly imprinted stationary phase prepared using racemic DNB as the template. A chiral monomer, (S)-(-)-N-methacryloyl-1naphthylethylamine, was utilized as the functional monomer toward the racemic template, and its chiral recognition ability was, interestingly, found to be enhanced through racemic molecular imprinting. A thermodynamic discussion briefly suggests that the observed chiral recognition ability of the racemic imprinting was proper value. Molecular imprinting is an easy and effective method to prepare polymeric media having specific molecular recognition toward the template utilized.1-3 Molecularly imprinted polymers (MIPs) are dominantly utilized as stationary phases in highperformance liquid chromatography (HPLC), adsorbents for solidphase extraction, and even reaction media.4 HPLC stationary phases prepared by the molecular imprinting technique with a chiral template result in chiral stationary phases, and those often show quite good chiral recognition ability.5-7 Although many examples of optically active molecular imprinting have been reported so far,8 we believe there is something inconsistent about chiral separation using current molecular imprinting methods. Actually, most of the examples are examined in terms of their chiral recognition ability using HPLC and the obtained chiral recognition ability is really great, but to prepare the molecularly imprinted chiral stationary phase, we have to use gram amounts of the isolated chiral template. This chiral compound will be chirally discriminated afterward by HPLC, and the separation factor will be evaluated. This is an inconsistency from the viewpoint of the requirement of chromatographic chiral separation of racemic solutes. In this study, we have examined an easily prepared, chiral functional monomer to realize the molecularly imprinted chiral (1) Sellergren, B.; Ekberg, B.; Mosbach, K. J. Chromatogr. 1985, 347, 1-10. (2) Shea, K. J.; Sasaki, D. Y. J. Am. Chem. Soc. 1991, 113, 4109-4120. (3) Wulff, G. ACS Symp. Ser. 1986, 308, 186-230. (4) Wulff, G. Angew. Chem., Int. Ed. Engl. 1995, 34, 1812-1832. (5) Vlatakis, G.; Andersson, L. I.; Mu ¨ ller, R.; Mosbach, K. Nature 1993, 361, 645-647. (6) Fischer, L.; Mu ¨ ller, R.; Ekberg, B.; Mosbach, K. J. Am. Chem. Soc. 1991, 113, 9358-9360. (7) Kempe, M.; Mosbach, K. J. Chromatogr. A 1994, 664, 276-279. (8) Kempe, M.; Mosbach, K. J. Chromatogr. A 1995, 694, 3-13. S0003-2700(97)00703-8 CCC: $15.00 Published on Web 01/27/1998
© 1998 American Chemical Society
stationary phase prepared using racemic template. Since a racemate is rarely more expensive than its enantiomer, the proposed method in this study is just a preliminary case but becomes a possible example for a practically applicable technique to prepare the chiral stationary phase by the molecular imprinting technique with racemic templates. EXPERIMENTAL SECTION N-(3,5-Dinitrobenzoyl)-R-methylbenzylamine (DNB) was utilized as the test template for this study and purchased from Aldrich, while (S)-(-)-methacryloyl-1-naphthylethylamine ((S)MNEA) was used as a functional monomer and prepared through a typical condensation reaction between commercially available (S)-1-naphthylethylamine and methacryloyl chloride, both of which were purchased from Aldrich. A cross-linking agent, ethylene dimethacrylate (EDMA), was purchased from Nacalai Tesque (Kyoto, Japan) and purified using a standard purification technique. All the solvents used for HPLC were of chromatographic grade and used as received. Molecularly imprinted polymer particles were prepared through a typical two-step swelling and polymerization method; therefore, the obtained particles are uniformly sized.9-11 Size uniformity of the polymer particles is very important in order to obtain reproducibility and easy operation. The molecularly imprinted polymer particles were prepared using 4.0 mL of EDMA, 4.0 mL of the porogen, toluene, and 2.0 mmol of the chiral functional monomer, (S)-(-)-MNEA, with chiral or racemic template, DNB (1.0 mmol). An unimprinted base stationary phase and an unimprinted chiral stationary phase with (S)-(-)-MNEA were also prepared as the reference stationary phases. RESULTS AND DISCUSSION Although discussions about chiral discriminations between DNB derivatives and naphthyl derivatives through π-π interaction have been reported with silica-based chiral stationary phases,12-14 the chiral functional monomer utilized in this study, (S)-MNEA (9) Smigol, V.; Svec, F.; Hosoya, K.; Wang, Q.; Fre´chet, J. M. J. Angew. Makromol. Chem. 1992, 195, 151-164. (10) Hosoya, K.; Yoshizako, K.; Tanaka, N.; Kimata, K.; Araki, T.; Haginaka, J. Chem. Lett. 1994, 1437-1438. (11) Ugelstad, J.; Kaggerud, K. H.; Hansen, F. K.; Berge, A. Makromol. Chem. 1979, 180, 737-744. (12) Oi, N.; Kitahara, H.; Aoki, F. J. Chromatogr. A 1995, 694, 129-134. (13) Veigl, E.; Bo¨hs, B.; Mandl, A.; Krametter, D.; Lindner, W. J. Chromatogr. A 1995, 694, 130-150. (14) Veigl, E.; Bo¨hs, B.; Mandl, A.; Krametter, D.; Lindner, W. J. Chromatogr. A 1995, 694, 151-161.
Analytical Chemistry, Vol. 70, No. 5, March 1, 1998 943
Figure 1. Chiral recognition ability of (S)-MNEA using copolymerization technique. Chromatographic conditions: mobile phase, hexane/ethyl acetate ) 1:1 (v/v); flow rate, 1.0 mL/min; detection, UV, 254 nm.
Figure 2. Chiral discrimination toward DNB on the racemic MIP. Chromatographic conditions: mobile phase, hexane/ethyl acetate ) 1:1 (v/v); flow rate, 1.0 mL/min; detection, UV, 254 nm; solute, (()DNB, 3.17 nmol.
Chart 1
(Chart 1), is not actually an effective or good chiral selector for the chiral amide compounds derived from R-methylbenzylamine if this monomer is just used in a copolymerization technique (not molecular imprinting), as shown in Figure 1. This is probably due to the fact that a cross-linked polymer is used as the base stationary phase instead of silica gels; therefore, chiral recognition ability is somehow different from those on silica-based chiral stationary phases.15 In addition, molecularly imprinted stationary phases prepared utilizing chiral (S)-DNB as the template with achiral functional monomers only gave 1.40-1.45 as the R values for DNB with poor resolutions. On the other hand, molecular imprinting using racemic template can be performed with racemic DNB, (()-DNB, and the chiral functional monomer (S)-MNEA with EDMA as cross-linking agent. Figure 2 shows a chromatogram of chiral discrimination toward DNB on the molecularly imprinted stationary phase (MIP) with the racemic template, namely “racemic MIP”. In this case, the observed separation factor R was 1.40, which is greater than that observed for the usual copolymerized stationary phase with (S)-MNEA as chiral comonomer (R ) 1.18, Figure 1) and also even comparable with those on the MIPs prepared utilizing chiral (S)-DNB as the template with achiral functional monomers (R ) 1.40-1.45). Although preferential interaction between (S)-DNB in the racemate and (S)-MNEA in the solution of the porogen and the monomers has not been elucidated yet, these facts suggest that, in the MIP, once the template fits into the molecularly imprinted binding sites, the chiral functional monomer utilized
can enhance chiral recognition ability.16 Even in a reversed-phase mode, 60% aqueous acetonitrile, a separation factor R ) 1.10 with moderate resolution was obtained on the racemic MIP, while almost no separation was found on the usual copolymer of (S)MNEA and EDMA. Another advantage of this racemic MIP is that separation of (4-nitrobenzoyl)-R-methylbenzylamine (NB) and DNB can be achieved under the chromatographic conditions employed, while no separation of NB and DNB is found with the base stationary phase. Although the peak shapes of the separated DNBs are relatively broad, only good chiral discrimination for DNBs was found, and other chiral amides, such as NB having one nitro group on the phenyl ring and B having no nitro group on the phenyl ring, were rarely or poorly separated on the racemic MIP, as shown in Figure 3. A thermodynamic relationship is established to evaluate the observed separation factor R value on the racemic MIP. Figure
(15) Hosoya, K.; Yoshizako, K.; Tanaka, N.; Kimata, K.; Araki, T.; Fre´chet, J. M. J. J. Chromatogr. A 1994, 666, 449-455.
(16) Hosoya, K.; Yoshizako, K.; Shirasu, Y.; Kimata, K.; Araki, T.; Tanaka, N.; Haginaka, J. J. Chromatogr. A 1996, 728, 139-147.
944 Analytical Chemistry, Vol. 70, No. 5, March 1, 1998
Figure 3. Separation of amide derivatives on the base stationary phase (left) and the racemic MIP (right). Chromatographic conditions are the same as those in Figure 2. Solutes, (()-DNB, 3.17 nmol; (()NB, 1.85 nmol; (()-B, 4.44 nmol.
Table 1. Calculated Separation Factors stationary phasea
Rcalcd
∆(∆G°)calcd (kcal mol-1)
Robsd
∆(∆G°)obsd (kcal mol-1)
symbol
(S)-MNEA (S)-MNEA + (S)-DNB (S)-MNEA + (R)-DNB (S)-MNEA + (()-DNB
1.16 2.79 1.65 1.37
-0.089 -0.617 -0.313 -0.192
1.18 2.74 1.69 1.40
-0.100 -0.606 -0.314 -0.203
∆(∆G°)ON ∆(∆G°)OS ∆(∆G°)OR ∆(∆G°)Ora
a (S)-MNEA was prepared using (S)-MNEA and no template by an usual copolymerization technique. (S)-MNEA + (S)-DNB and (S)-MNEA + (R)-DNB were prepared using (S)-MNEA as the host monomer and as template, (S)-DNB or (R)-DNB, respectively. (S)-MNEA + (()-DNB was prepared using (S)-MNEA and racemic DNB as the template.
4 shows schematically the relationship set for this study. Since unimprinted stationary phase, “BASE”, still has retentivity toward DNBs in the employed mobile phase, these relationships might not be strictly correct. However, we use these relationship only to compare relative R values of all the prepared stationary phases. Based on eq 1 and other constant values, four equations can be written.
∆(∆G°) ) -RT ln R (kcal/mol)
(1)
R ) 8.314 J/(K‚mol) T ) 303 K (at 30 °C) 1 kJ/mol ) 0.238 kcal/mol ∆(∆G°)NS ) ∆(∆G°)Nra - ∆(∆G°)NR
(2)
∆(∆G°)OS ) ∆(∆G°)ON + ∆(∆G°)NS
(3)
∆(∆G°)NR ) ∆(∆G°)OR - ∆(∆G°)ON
(4)
∆(∆G°)Ora ) ∆(∆G°)ON + ∆(∆G°)Nra
(5)
These equations can be converted using experimentally obtainable values.
∆(∆G°)OS ) ∆(∆G°)ON + ∆(∆G°)Ora - ∆(∆G°)OR (6) ∆(∆G°)OR ) ∆(∆G°)ON + ∆(∆G°)Ora - ∆(∆G°)OS (7) ∆(∆G°)Ora ) ∆(∆G°)OS + ∆(∆G°)OR - ∆(∆G°)ON (8) ∆(∆G°)ON ) ∆(∆G°)OS + ∆(∆G°)OR - ∆(∆G°)Ora (9) Using these converted equations, we calculated separation factors on the four types of stationary phases. These values are listed in Table 1. As shown in Table 1, relatively good agreements are obtained between the calculated values and observed values. These facts might suggest that the observed separation factor on
Figure 4. Schematic thermodynamic relationship. Arrows having the same symbols have the same length.
the racemic MIP prepared in this study is a proper value. In addition, chiral recognition ability of (S)-MNEA on the racemic MIP is found to be enhanced through racemic molecular imprinting compared with stationary phase (S)-MNEA, where no template is utilized. CONCLUSION Racemic MIP can be prepared and shows a chiral discrimination toward DNB. The observed separation factor on the racemic MIP is thought to be a proper value based on relative thermodynamic calculations, and the chiral recognition ability of (S)-MNEA is enhanced through molecular imprinting. This work should be a special case and improved in terms of resolution and peak shape; however, racemic MIP should become a more practical method to prepare a chiral stationary phase using the current molecular imprinting technique. The improvement and another application as well as elucidation of the mechanism for formation of biding sites using racemic template and chiral functional monomer are in progress to show the generality of the method. ACKNOWLEDGMENT This work is partly supported by the funds from the Japanese Ministry of Education (Nos. 09640726 and 09044081). Received for review July 3, 1997. Accepted December 9, 1997. AC9707038
Analytical Chemistry, Vol. 70, No. 5, March 1, 1998
945
