Anal. Chem. 1999, 71, 2844-2849
Color Change Indicators for Molecules Using Methyl Red-Modified Cyclodextrins Tetsuo Kuwabara,*,† Hiroki Nakajima,† Masato Nanasawa,† and Akihiko Ueno*,‡
Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Yamanashi University, 4 Takeda, Kofu 400-8511, Japan, and Department of Bioengineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan
β-Cyclodextrin derivatives, MRCD and p-MRCD, which have a 4-(dimethylamino)azobenzene moiety with a carbonyl substituent at 2′ and 4′ positions, respectively, have been prepared as color change indicators for detecting organic compounds. In a 10% ethylene glycol solution, MRCD and p-MRCD form intramolecular self-complexes in which the pendant dye moiety is included in the cyclodextrin cavity with an orientation parallel and perpendicular to the cyclodextrin axisis, respectively. When guest molecules are added to the acidic solutions of MRCD (pH 1.60) and p-MRCD (pH 2.40), they exhibit color changes from yellow to red for MRCD and from orange to red for p-MRCD. These color changes, which arise from the structural change of the dye moieties from the azo form to the azonium one, are caused when MRCD and p-MRCD undergo a conformational change in which the dye moieties inserted in the cyclodextrin cavities are excluded to outside of the cavities upon guest accommodation. The extent of the guest-induced color changes of MRCD and p-MRCD depend on the shape, size, number, and position of the functional group of guest molecules. Selectivities between MRCD and p-MRCD in guest detection are roughly parallel and reflected in the host-guest binding constants. Among guest molecules examined, ursodeoxycholic acid and chenodeoxycholic acid were detected by MRCD and p-MRCD with high sensitivities. 1-Adamanetanecarboxylic acid and (-)borneol were also detected with high sensitivities. In neutral conditions, however, the selectivity in guest detection of p-MRCD is different from that in acidic conditions as shown by the fact that, for example, 1-adamantanol and 2-adamantanol were detected by p-MRCD with larger sensitivities than 1-adamanetanecarboxylic acid. The result indicates that the ionic nature of the guest molecules is an important factor for detection of the guest molecules. All these results demonstrate that MRCD and p-MRCD can be used as color change indicators for detecting various organic compounds in aqueous solution. During the past decade, much effort has been devoted to developing artificial receptors that transform binding of chemical † ‡
Yamanashi University. Tokyo Institute of Technology.
2844 Analytical Chemistry, Vol. 71, No. 14, July 15, 1999
species into spectroscopic signals. The signal transduction of these systems usually occurs on the basis of host-guest complexation. Some examples for artificial ionophores based on crown ethers, cryptands, or calixarenes have already been reported.1 In these systems, absorption or fluorescence properties of the host molecules were perturbed by binding of metal or ammonium cations into the cavity of the macrocyclic part of the hosts. In contrast to these, there have been few examples for receptors that are responsive to neutral organic molecules.2-7 Cyclodextrins (CDs), which are cyclic oligomers of D-glucose, can form inclusion complexes with a wide variety of neutral organic species in aqueous solution.8,9 Although CDs are spectroscopically inert, they can be converted into spectroscopically active hosts by modification with an appropriate chromophore. Ueno et al. discovered that CDs can be used as starting materials for constructing fluorescent indicators for organic molecules and they have shown that modified CDs bearing fluorophores such as pyrene,10,11 naphthalene,12,13 dansyl,14-17 or N,N-dimethylaminobenzoyl18,19 moieties can be used as sensors for detecting (1) Comprehensive Supramolecular Chemistry; Gokel, G. W., Ed.; Pergramon: Oxford, 1996; Vol. 1. (2) Inouye, M.; Kim, K.; Kitao, T. J. Am. Chem. Soc. 1992, 114, 778-780. (3) Bernardo, A. R.; Stoddart, J. F.; Kaifer A. E. J. Am. Chem. Soc. 1992, 114, 10624-10631. (4) Inouye, M.; Hashimoto, K.; Isagawa, K. J. Am. Chem. Soc. 1994, 116, 55175518. (5) Inouye, M.; Miyake, T.; Furusyo, M.; Nakazumi, H. J. Am. Chem. Soc. 1995, 117, 12416-12425. (6) Koh, K. N.; Araki, K.; Ikeda, A.; Otsuka, H.; Shinkai, S. J. Am. Chem. Soc. 1996, 118, 755-758. (7) Kudo, Y.; Kaeda, S.; Tokita, S.; Kudo, M. Nature 1996, 382, 522-524. (8) Comprehensive Supramolecular Chemistry; Szejtli, J., Osa, T., Eds.; Pergramon: Oxford, 1996; Vol. 3. (9) Szejtli, J. Cyclodextrins and Their Inclusion Complexes; Akademiai Kindo: Budapest, 1982. (10) Ueno, A.; Suzuki, I.; Osa, T. Anal. Chem. 1990, 62, 2461-2466. (11) Suzuki, I.; Sakurai, Y.; Ohkubo, M.; Ueno, A.; Osa, T. Chem. Lett. 1992, 2005-2008. (12) Ueno, A.; Minato, S.; Osa, T. Anal. Chem. 1992, 64, 1154-1157. (13) Ueno, A.; Minato, S.; Osa, T. Anal. Chem. 1992, 64, 2562-2565. (14) Ikeda, H.; Nakamura, M.; Ise, N.; Oguma, N.; Nakamura, A.; Ikeda, T.; Toda, F.; Ueno, A. J. Am. Chem. Soc. 1996, 118, 10980-10988. (15) Ikeda, H.; Nakamura, M.; Ise, N.; Nakamura, A.; Ikeda, T.; Toda, F.; Ueno, A. J. Org. Chem. 1996, 62, 1411-1418. (16) Wang, Y.; Ikeda, T.; Ikeda, H.; Ueno, A.; Toda, F. Bull. Chem. Soc. Jpn. 1994, 67, 1598-1607. (17) Wang, Y.; Ikeda, T.; Ueno, A.; Toda, F. Chem. Lett. 1992, 863-866. (18) Hamasaki, K.; Ueno, A.; Toda, F.; Suzuki, I.; Osa, T. Bull. Chem. Soc. Jpn. 1994, 67, 516-523. (19) Hamasaki, K.; Ikeda, H.; Nakamura, A.; Ueno, A.; Toda, F.; Suzuki, I.; Osa, T. J. Am. Chem. Soc. 1993, 115, 5035-5040. 10.1021/ac9814041 CCC: $18.00
© 1999 American Chemical Society Published on Web 06/08/1999
organic compounds by guest-induced variation in the emission intensity in aqueous solution. In these CD-based sensors, the binding of guest species is transduced into fluorescent signals through the guest-induced locational change of the chromophores, mostly from inside to outside of the CD cavity. On the other hand, recently, some dye-modified CDs have been prepared as new series of indicators for molecules. p-Nitrophenol-,20,21 alizarine yellow-,22 and phenolphthalein-modified CDs23,24 have been reported as absorption change sensors for molecules, each acting in neutral or alkaline solution. Previously we reported that methyl red-modified CD25 (MRCD) acts as a color change sensor in acidic solution. Here we report the sensor ability of p-methyl red-modified CD (p-MRCD), which is structural isomer of MRCD, as compared with that of MRCD. The sensor ability reflects the structural differences in the two sensor molecules. EXPERIMENTAL SECTION Materials. MRCD and p-MRCD were prepared according to the procedure reported previously.26 1H NMR and elemental analyses were satisfactory. The guest compounds were commercially guaranteed reagents and used without further purification. Measurements. Due to the poor solubility of MRCD and p-MRCD in pure water, 10% ethylene glycol aqueous solutions of MRCD (0.03 mM) and p-MRCD (0.015 mM) were used. The solutions were made by addition of water into an ethylene glycol solution of MRCD (0.3 mM) or p-MRCD (0.15 mM). Hydrochloric acid was used to make the pH 1.60 solution of MRCD and pH 2.40 solution of p-MRCD, and phosphate buffer was used to prepare the pH 7.40 solution of p-MRCD. The pH of the solution (20) Matsushita, A.; Kuwabara, T.; Nakamura, A.; Ikeda, H.; Ueno, A. J. Chem. Soc., Perkin Trans. 2 1997, 1705-1710. (21) Kuwabara T.; Matsushita, A.; Nakamura, A.; Ueno, A.; Toda, F. Chem. Lett. 1993, 2081-2084. (22) Aoyagi, T.; Nakamura, A.; Ikeda, H.; Ikeda, T.; Mihara, H.; Ueno, A. Anal. Chem. 1997, 69, 659-663. (23) Kuwabara T.; Takamura M.; Matsushita, A.; Ueno, A.; Toda, F. Supramol. Chem. 1996, 8, 13-15. (24) Kuwabara T.; Takamura M.; Matsushita, A.; Ikeda, H.; Ueno, A.; Toda, F. J. Org. Chem. Soc. 1998, 63, 8729-8735. (25) Ueno, A.; Kuwabara, T.; Nakamura, A.; Toda, F. Nature 1992, 356, 136137. (26) Kuwabara, T.; Nakamura, A.; Ueno, A.; Toda, F. J. Phys. Chem. 1994, 98, 6297-6303.
Figure 1. Absorption spectra of MRCD (3.0 × 10-5 M) and p-MRCD (1.5 × 10-5 M) at pH 6.80 (- - -) and 1.00 (s) in a 10% ethylene glycol aqueous solution.
was measured on a Beckmann 30 pH meter, which was calibrated at 25 °C with pH standard solutions of pH 4.01 and 6.86. Absorption spectra and induced circular dichroism spectra were recorded on Shimadzu UV-3100 and 160 spectrophotometers and a Jasco J-600 spectropolarimeter at 25 °C, respectively. The value of ∆I/I0 was used as sensitivity parameter for estimating sensor ability of MRCD and p-MRCD, where ∆I ) I - I0 and I0 and I are the absorption intensities of the hosts, alone and in the presence of a guest, respectively. The ∆I/I0 values for a variety of guests were obtained at 510 nm for MRCD and p-MRCD in the acidic or neutral medium. The host-guest binding constants (K) of MRCD and p-MRCD for various guests were determined by the variations of absorption intensity with the aid of nonlinear curve-fitting analysis based on the following equation:
∆I ) ∆Imax[(G0 + H0 + 1/K) - {(G0 + H0 + 1/K)2 - 4G0H0}1/2] 2H0 where H0 and G0 represent the initial concentrations of host and guest, respectively. The difference in the absorption intensity at 510 nm between the host alone and in the presence of guest is expressed as ∆I value. The concentration of the complex is reflected in the magnitude of ∆I. When the hosts exist totally as the inclusion complexes, ∆I is equal to ∆Imax. RESULTS AND DISCUSSION Spectral Changes of MRCD and p-MRCD. Figure 1 shows the absorption spectra of MRCD and p-MRCD in neutral and acidic conditions. As stated previously,26 the absorption bands of MRCD and p-MRCD around 450 nm in the neutral condition indicate that their dye moieties exist as the azo form. In the acidic condition, Analytical Chemistry, Vol. 71, No. 14, July 15, 1999
2845
Figure 2. Induced circular dichroism spectra of MRCD (s) and p-MRCD (- - -) at pH 6.80 in a 10% ethylene glycol aqueous solution.
however, MRCD and p-MRCD exhibit different absorption patterns with the absorption maximums at 320 and 510 nm for MRCD and p-MRCD, respectively, suggesting that MRCD and p-MRCD exist as the ammonium form and the azonium form, respectively, being protonated at the dimethylamino group for MRCD and at the azo group for p-MRCD in the dye moiety. This is reflected in the different inclusion patterns between MRCD and p-MRCD, which are confirmed from the induced circular dichroism spectra of MRCD and p-MRCD (Figure 2).26 The positive and negative dichroism bands for MRCD were observed around 420 and 470 nm, respectively, each ascribing to the π-π* and n-π* transition of the dye moiety, while the negative and the positive bands were observed for p-MRCD around 420 and 470 nm, respectively. These results indicate that the dye moiety of MRCD and p-MRCD is inserted into its β-CD cavity with an orientation parallel and perpendicular to the CD axis, respectively. Since the protonation for azo dyes usually occurs on the azo group,27 the protonation occurring on the dimethylamino group of the dye moiety of MRCD is due to the deep inclusion of the dye moiety into the hydrophobic CD cavity. This marked difference in the absorption and the dichroism behavior observed for MRCD and p-MRCD should arise from the fact that MRCD and p-MRCD have the amide linkage at different positions with respect to the azo group in the dye units. Figure 3 shows the absorption spectra of MRCD and p-MRCD, alone and in the presence of 1-adamanatanecarboxylic acid (1ACA) as the guest. Upon addition of 1-ACA, MRCD changed color from yellow to red at pH 1.60 with the shift of the absorption maximum from 455 to 510 nm, while p-MRCD changed from orange to red at 2.40 with the shift from 485 to 510 nm. These spectral changes indicate that MRCD and p-MRCD change the location of the dye moiety upon host-guest complexation. The dye moieties of MRCD and p-MRCD, each existing as the azo form under these conditions, are accommodated in their CD cavities with the axial and the equatorial orientations for MRCD and p-MRCD, respectively, and are excluded to the outside of cavities upon guest binding, resulting in the protonation of the azo group in the dye moieties. These were confirmed from their dichroism spectra. The intensities of the positive bands around 420 and 480 nm for MRCD and p-MRCD decreased upon guest addition, suggesting the dye moieties undergo the locational or (27) Buva´ri, A.; Barcza, L. J. Inclusion Phenom. Mol. Recognit. Chem. 1989, 7, 313-319.
2846 Analytical Chemistry, Vol. 71, No. 14, July 15, 1999
Figure 3. Absorption spectra of MRCD at pH 1.60 and p-MRCD at pH 2.40 and pH 7.40, alone (- - -) and in the presence of the 1-ACA (s) in a 10% ethylene glycol aqueous solution. The solutions of MRCD at pH 1.60, p-MRCD at pH 2.40, and p-MRCD at pH 7.40 contained 0.6, 0.06, and 0.45 mM 1-ACA, respectively.
orientational change, weakening the interaction with the CD cavity. On the other hand, p-MRCD showed a guest-induced slight red shift even at neutral pH where MRCD showed no change in absorption spectra. This is due to the conformational change of p-MRCD upon guest binding. The dye moiety interacting with the hydrophobic CD cavity is excluded to the bulk water environment. On the other hand, MRCD has no guest binding ability under neutral conditions due to the tight inclusion of the dye moiety in its CD cavity. The other guests examined here caused similar spectral changes, although the extents of the variations were different. Isosbestic points were observed for every host-guest combination, indicating that the complexation proceeds solely based on 1:1 host-guest stoichiometry. The modified CDs whose spectral variations are affected by the presence of the guest molecules can be useful as host-guest sensors for detecting organic species in solution, and we have examined the abilities of MRCD and p-MRCD as such sensors. Response at Constant Concentration. A variety of organic compounds shown in Table 1 were used to examine the sensor abilities of MRCD and p-MRCD. The structures of the guest are
Table 1. Guest-Induced Variation of the Sensitivity Factor (∆I/I0) of MRCD and p-MRCD and Host-Guest Binding Constants (K) in a 10% Ethylene Glycol Aqueous Solutiona p-MRCD MRCD, pH 1.60 guest (-)-borneol (BN) (+)-camphor ((+)-CP) (-)-camphor (-)-CP) (+)-menthol ((+)-MT) (-)-menthol ((-)-MT) nerol (NR) geraniol (GR) cyclohexanol (CHN) cyclooctanol (CON) 1-adamanatanol (1-AN) 2-adamanatanol (2-AN) 1-adamanatanecarboxylic acid (1-ACA) 1-adamanataneamine (1-AA) cholic acid (CA) deoxycholic acid (DCA) chenodeoxycholic acid (CDCA) ursodeoxycholic acid (UDCA) corticosterone (CCTR) a
∆I/I0 0.164b 0.0190c 0.0252b 0.0373b 0.0252b 0.0297b 0.0025b 0.0084b 0.0013b 0.0381b 0.411b 0.0501c 0.404b 0.053c 1.376b 0.261c 0.0360b 0.0037c 0.0300c 0.0478c 0.658c 1.503c 0.0138c
pH 2.40
K 230 39 68 75 64 d d 17 35 730
∆I/I0 0.751b 0.192c 0.427b 0.399b 0.264b 0.259b 0.129b 0.139b 0.149b 0.334b
pH 7.40 K 7970 1390 1600 1240 1210 460 460 890 1600 26600
0.380c 1270
59000 0.524c
5550 93 900 4130 12400 77100 550
∆I/I0
K
0.212b
2080
0.0286c 0.0566b 0.0619b 0.0301b 0.0610b 0.0241b 0.0195b 0.0074b 0.0501b 0.302b 0.0409c 0.304b 0.0485c
250 270 140 530 d d 23 410 5270 7540
104400 0.783c 0.395b 0.0559c 0.122c 0.150c 0.644c 0.818c 0.0437c
1680 4540 d 52900 1110000 1480
980 0.0240c 0.0977b 0.0233c 0.0188c 0.0194c 0.0271c 0.0441c 0.0188c
770 94 160 2110 6320 83
MRCD, 3.0 × 10-5 M; p-MRCD, 1.5 × 10-5 M, the values of ∆I and I0 at 510 nm were used. b 0.3 mM. c 0.03 mM. d Not obtained.
Chart 1
Chart 3
Chart 2
guests, p-MRCD showed a moderate response with sensitivities from 0.129 to 0.427, which are much larger than those of MRCD, whose values range from 0.0025 to 0.0373. This fact indicates that p-MRCD shows responses with higher sensitivity to the presence of the guests than MRCD. However, the selectivities of MRCD and p-MRCD for guest detection are roughly parallel. Both hosts detected (+)- and (-)-camphor (CP), which have the same bicyclo[2, 2, 1]heptane framework as that of (-)-BN, and monocyclic (+)- and (-)-menthol (MT), with relatively high responses. In contrast to these, nerol (NR) and geraniol (GR), both being acyclic monoterpenes with cis and trans forms, respectively, were detected with smaller sensitivities. These results indicate that the roundlike molecular shape of the guest is suited for detection by MRCD or p-MRCD with high sensitivity. As a result, the orders of sensitivities are (-)-BN . (+)-CP g (-)-CP > (+)-MT g (-)MT > GR g NR and (-)-BN . (-)-CP > (-)-MT g (+)-CP ) (+)-MT . GR g NR for p-MRCD and MRCD, respectively. Although acyclic NR and GR were not easily detected by MRCD,
shown in Charts 1-3. Among terpenoid compounds whose structures are shown in Chart 1, (-)-borneol ((-)-BN) was detected by MRCD at pH 1.60 and by p-MRCD at pH 2.40 with the highest sensitivities of 0.164 and 0.751, respectively. The result is consistent with the fact that the binding constants of MRCD and p-MRCD for (-)-BN are 230 and 7970 M-1, respectively, and the values are much larger than those for other guests. For other
Analytical Chemistry, Vol. 71, No. 14, July 15, 1999
2847
MRCD is useful for detecting (-)-BN among the terpenoid compounds because the sensitivity of MRCD for (-)-BN was 4.46.5 times higher than other cyclic terpenoid guests, while that for p-MRCD was 1.8-2.9 times higher. Such similar guest dependency of MRCD and p-MRCD and smaller ∆I/I0 values of MRCD are consistent with the binding constants of MRCD and p-MRCD. The binding constants of p-MRCD and MRCD range from 1210 to 1600 M-1 and from 39 to 75 M-1 for (+)-CP, (-)CP, (+)-MT, and (-)-MT, respectively. In the case of acyclic compounds, NR and GR, the binding constant is 460 M-1 for p-MRCD while it could not be obtained for MRCD owing to the minimal spectral changes. The binding constants of p-MRCD are ∼20 times larger than those of MRCD and the orders of the binding constants of MRCD and p-MRCD are roughly parallel. The smaller binding ability of MRCD may be due to the tight inclusion of the dye moiety of MRCD in its CD cavity. In this case, the dye moiety of MRCD may act like an intramolecular inhibitor for guest binding. The sensor ability of p-MRCD was also examined at pH 7.40 in the presence of guest (0.3 mM). The highest sensitivity values of p-MRCD were observed for (-)-BN with a ∆I/I0 value of 0.212. Other guests show moderate responses with the sensitivities from 0.0195 to 0.0619. The values obtained at pH 7.40 are smaller than those obtained at pH 2.40, although the guest dependency of p-MRCD at pH 7.40 is similar to the case at pH 2.40 with the order (-)-BN . (-)-CP g (-)-MT > (+)-CP g (+)-MT > NR g GR. This suggests that it is better to use p-MRCD as a sensor in the acidic medium rather than in the neutral one. The binding constant of p-MRCD for (-)-BN at pH 7.40 is 2080 M-1, and the binding constants for other guests range from 140 to 530 M-1 except for NR and GR, whose binding constants could not be obtained because of the poor solubility. The binding constants of p-MRCD at pH 7.40 were smaller than those at pH 2.40 but larger than those of MRCD at pH 1.60. It seems that the dye moiety of p-MRCD may not act as an inhibitor for guest binding even in the neutral medium. Among cyclic alcohols, cyclooctanol (CON) was detected by MRCD and p-MRCD with higher sensitivity than cyclohexanol (CHN) in both acidic and neutral conditions, indicating that the cyclooctyl framework is a better size to fit in the β-CD cavity than the cyclohexyl. It is noted, furthermore, that the binding constants of MRCD and p-MRCD for CON are ∼2 times higher than for CHN in the acidic medium and the binding constant of p-MRCD for CON, in the neutral medium, is 18 times higher than that for CHN. This result suggests that the neutral condition is better than the acidic for detecting CON. On the other hand, among the adamantane derivatives, 1-ACA was detected by MRCD and p-MRCD with highest sensitivities of 0.261 and 0.783 in the acidic medium, respectively, at a guest concentration of 0.03 mM. 1-Adamantaneamine (1-AA), which has an amino group in place of the carboxylic acid group of 1-ACA, was detected with lower sensitivities than 1-adamantanol (1-AN) and 2-adamantanol (2-AN). The small ∆I/I0 value obtained for 1-AA may be due to its ionic nature, which seems unfavorable for it to be inserted in the hydrophobic CD cavity, because 1-AA has a positively charged ammonium cation in the acidic medium. Such charge effect of the guest on guest binding ability was observed under neutral conditions. At pH 7.40, 1-ACA was detected by p-MRCD with 2848 Analytical Chemistry, Vol. 71, No. 14, July 15, 1999
smaller sensitivity than 1-AN and 2-AN because 1-ACA has a negative charge at this pH. 2-AN showed higher sensitivity than 1-AN for both hosts. For adamantane derivatives, the sensitivity of MRCD and p-MRCD in acid is the same with the order 1-ACA > 2-AN > 1-AN > 1-AA, while that in the neutral condition is 2-AN > 1-AN > 1-ACA g 1-AA. These results imply that the ionic nature as well as the shape of the guest molecule is important for detecting the guest species. The highest binding constant of p-MRCD was achieved by 1-ACA with the value of 104 400 M-1 in the acidic medium, followed by 2-AN, 1-AN, and 1-AA. A similar trend was observed for MRCD in the acidic medium with smaller binding constants than those of p-MRCD. This result also suggests that p-MRCD is a better host for detecting the guests in acidic medium. At pH 7.40, 1-AN and 2-AN showed larger binding constants than the other charged guests. Table 1 shows the ∆I/I0 values of MRCD and p-MRCD for the steroid compounds. The structures of the guests examined are shown in Chart 3. The ∆I/I0 values of MRCD and p-MRCD were obtained at the guest concentration of 0.03 mM. Corticosterone (CCTR) and cholic acid derivatives such as cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), and ursodeoxycholic acid (UDCA) were detected by MRCD and p-MRCD with a sensitivity order of UDCA > CDCA > DCA > CA > CCTR in both acidic and neutral conditions, although the values of p-MRCD at pH 7.40 were smaller than those at acidic pH. CDCA and UDCA are isomers with the stereochemical difference of one hydroxyl at the C7 position, while DCA is a regioisomer of CDCA and UDCA with one hydroxyl at the C12 position in place of C7 of the steroidal framework. Therefore, the different sensitivities observed for DCA, CDCA, and UDCA imply the remarkable molecular recognition abilities of MRCD and p-MRCD. CA, which has one more hydroxyl group than DCA, CDCA, and UDCA, was detected by MRCD and p-MRCD with lower sensitivity. CCTR, which is 20-keto steroid, was not well detected by MRCD and p-MRCD. These results are consistent with their binding constants. UDCA showed the highest binding constants for MRCD and p-MRCD, their values being 77 100 M-1 for MRCD and 1 110 000 M-1 for p-MRCD, followed by CDCA with the values of 12 400 M-1 for MRCD and 52 900 M-1 for p-MRCD. Under the neutral conditions, the binding constants of p-MRCD are much smaller than the those in the acidic medium; being 6320 M-1 for UDCA and 2110 M-1 for CDCA. Response Ranges and Limit Concentrations of Detection. Figure 4 shows the ∆I/I0 values of MRCD and p-MRCD as a function of the guest concentration for (-)-BN, CHN, and CDCA. The value of MRCD increases with increasing guest concentration in the range of 10-5-10-4, 10-3-10-2, and 10-2 M for CDCA, (-)BN, and CHN, respectively. The results indicate that CDCA can be detected by MRCD even if the solution contains a considerable amount of BN or CHN, for example, 10-4 M BN or 10-3 M CHN. On the other hand, the value ∆I/Io of p-MRCD at pH 2.40 increases with increasing guest concentration of 10-5-10-4, 10-410-3, and 10-3 M for CDCA, (-)-BN, and CHN, respectively, indicating that p-MRCD responds to (-)-BN and CHN with a smaller concentration of guest than is the case for MRCD, although there is no change in the response range to CDCA. At pH 7.40, however, the ranges of guest concentration of p-MRCD were shifted to the more concentrated side as compared to those
Table 2. Limit Concentration (M) of Detection of MRCD and p-MRCD p-MRCD guest BN 1-AN 1-ACA UDCA CDCA
MRCD, pH 1.60 10-5
7.9 × 4.4 × 10-5 7.5 × 10-6 8.9 × 10-7 6.3 × 10-7
pH 2.40 10-5
4.3 × 1.5 × 10-5 3.4 × 10-6 4.3 × 10-6 2.1 × 10-6
pH 7.40 1.8 × 10-5 1.1 × 10-4 6.8 × 10-5 6.2 × 10-5 6.6 × 10-5
detection might be given by the guest having the larger binding constant with the host. Despite the smaller binding constant of MRCD, however, the limit concentration of detection of MRCD is lower than that of p-MRCD. This may be due to the larger standard deviation of p-MRCD as compared to MRCD. The larger standard deviation of p-MRCD is associated with the pH-sensitive absorbance of p-MRCD at 510 nm as compared to that of MRCD: the absorbance changing from 0.222 to 0.245 with MRCD while that of p-MRCD changed from 0.173 to 0.623 when the pH of the solution changed from 6.80 to 1.00, although the concentration of MRCD is 2 times higher than that of p-MRCD.
Figure 4. Sensitivity factor (∆I/I0) of MRCD and p-MRCD as a function of the concentration of (-)-BN (gray circle), CHN (O), and CDCA (b). The values of ∆I and I0 at 510 nm were used.
at pH 2.40. Especially, a marked shift in the range for CDCA is observed and its range is similar to that of (-)-BN. This indicates that p-MRCD acts as a sensor with higher sensitivity in acidic medium than in neutral medium. The above results suggest that a particular guest molecule having a large binding constant is detected by MRCD and p-MRCD with high selectivity in the presence of the other guests that have smaller binding constants. At the present time, however, distinguishing a particular molecule in a mixture of several compounds has limit because of the limitations of selectivity of the host compounds. Finally, the limit concentrations detectable by the absorption change of MRCD and p-MRCD were estimated from 2 times the standard deviation values for each condition. Table 2 shows the limit concentrations of detection of MRCD and p-MRCD for (-)BN, 1-AN, 1-ACA, CDCA, and UDCA. All hosts showed the lower limit concentrations of detection for CDCA and UDCA and the values of MRCD at pH 1.60 are the smallest for the guests. p-MRCD showed a lower limit concentration in acidic medium than in neutral. These results coincide with those obtained in the previous section. It is noted that the lower limit concentration of
CONCLUSION MRCD and p-MRCD, both having a dye moiety, change their color from yellow to red and from orange to red, respectively, by the addition of a guest in the acidic medium. The principle of the action is that the dye moiety included in the CD cavity is displaced by the guest molecules. The color changes of MRCD and p-MRCD are associated with the changes in locus of the dye moieties. The guest binding properties, however, were markedly affected by the structural difference of MRCD and p-MRCD as shown by the fact that the binding constants of p-MRCD are larger than those of MRCD in the acidic medium. Furthermore, MRCD loses the color change ability in neutral medium, but p-MRCD still shows a guestinduced slight red shift in absorption maximum. MRCD and p-MRCD act as better sensors for molecules in acidic condition than in neutral. Many color change indicators for detecting various organic compounds will be constructed on the same principle. ACKNOWLEDGMENT We are indebted to Nihon Shokuhin Kako for gifts of β-CD and one of the authors would like to thank professor Yoshihito Ikariyama and Dr. Shigeru Toyama of National Rehabilitation Center for the Disabled for their kind support. This work was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science Sports and Culture of Japan and the TV Yamanashi Science Development Fund. Received for review December 18, 1998. Accepted April 15, 1999. AC9814041
Analytical Chemistry, Vol. 71, No. 14, July 15, 1999
2849
