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Study on the interaction of methylene blue with cyclodextrin derivatives by absorption and fluorescence spectroscopy
Spectrochimica Acta Part A 59 (2003) 2935 /2941 www.elsevier.com/locate/saa
Study on the interaction of methylene blue with cyclodextrin derivatives by absorption and fluorescence spectroscopy Guomei Zhang, Shaomin Shuang *, Chuan Dong, Jinghao Pan Department of Chemistry, Shanxi University, Shanxi, Taiyuan 030006, China Received 18 December 2002; accepted 5 February 2003
Abstract The ability of b-cyclodextrin (b-CD), hydroxypropyl-b-cyclodextrin (HP-b-CD), and carboxymethyl-b-cyclodextrin (CM-b-CD) to break the aggregate of the methylene blue (MB) and to form 1:1 inclusion complexes has been studied by absorption and fluorescence spectroscopy. Experimental conditions including concentrations of various cyclodextrins (b-CD, HP-b-CD and CM-b-CD) and media acidity were investigated for the inclusion formation in detail. The formation constants are calculated by using steady-state fluorimetry, from which the inclusion capacity of different cyclodextrins (CDs) is compared. The results suggest that the charged b-cyclodextrin (CM-b-CD) is more suitable for inclusion of the cationic dye MB than the neutral b-cyclodextrins (b-CD, HP-b-CD) at pH/5. A mechanism is proposed which is consistent with the stronger binding of MB with CM-b-CD compared with the other CDs at pH/5. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Cyclodextrin; Methylene blue; Fluorescence
1. Introduction A wide variety of application of the phenothiazine dyes have been reported, for example, as sensitizers in solar energy conversion [1], redox mediators in catalytic oxidation reactions [2], active species in electrochromism [3] and dye lasers [4], ingredients in pharmaceutical preparation [5], and candidates for cancer therapy by intercalating between DNA base layers [6]. However, all of these applications are often complicated due to the
* Corresponding author. Fax: /86-351-701-1322. E-mail address: [email protected] (S. Shuang).
dimerization of dye molecules in aqueous media such as the great decrease of light sensitivity [7], which results from increased inner conversion of dimer exciter state. Thus, it is an important clue to improve the sensitivity of dyes towards obtaining a great number of monomers. Cyclodextrins (CDs) is one of the most important host molecules in supramolecular chemistry. CDs have the peculiar ‘interior hydrophobic, exterior hydrophilic’ structure forming a 1:1 or 1:2 inclusion complex with gust molecules, thus the physical, chemical and biochemical characters of guest molecules are modified [8,9]. The property of CDs can be used to control the position of monomer/dimer equilibrium and aggregation be-
1386-1425/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1386-1425(03)00123-9
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havior of dye, affecting electronic absorption and fluorescence spectra and photochemical and electrochemical properties of dyes. Different inclusion models have been observed, e.g. inclusion within the cavity or binding to the rim or penetrating partially into the CDs. CDs with inclusion properties have been widely used in pharmaceutical [10], food [11], environmental protection analysis [12], and enzyme modeling [13]. Methylene blue (MB) is one of phenothiazine dyes with a planar structure. It is today a generally accepted concept that planar dye molecules, such as acridine dyes, bind to DNA ‘intercalation’ mode. The interaction of planar molecules with DNA focuses on the aspects of biological probes and in the design of the efficient drugs targeted to DNA. Many analytical methods have been reported for the investigation on the complex of CD with MB including electrochemical method [14,15] and UV /Vis spectrotometry [16,17], etc. The recent methods are limited to parent b-CD. The purpose of this paper is to study the complexing behavior of MB with charged CD derivative CM-b-CD and neutral b-CD and HP-bCD by absorption and stead-state fluorescence spectroscopy technique. The complexing capacity of CDs with MB was compared. The possible inclusion mechanism was proposed that the charged CD was different from that of the neutral CDs.
analytical-reagent without purification. Double distilled water was used throughout. 2.2. Apparatus The absorption and fluorescence measurements were performed with a UV-265 spectrophotometer (Shimadzu, Japan), and a F-4500 spectrofluorimeter (Hitachi, Japan), respectively. Excitation and emission bandwidths were both set at 5 nm. pHs-2 meter was made in the 2nd Instrument Factory of Shanghai in China. All experiments were carried out at 209/1 8C. 2.3. Procedures A 1 ml aliquot of the stock solution (1.0 /10 4 mol/l) of MB was transferred into a 10 ml volumetric flask, then appropriate amount of 0.01 mol/l b-CD (or HP-b-CD or CM-b-CD) solution was added. The pH was controlled by 0.5 mol/l phosphate buffer solution. The mixed solution was diluted to final volume with distilled water and shaken thoroughly, following equilibrated for 30 min at 20 8C. All the measurements of absorption, fluorescence were made against the blank solution treated in the same way without CDs by using 1.0 cm quartz cell.
3. Results and discussion 2. Experimental 2.1. Reagents MB was of analytical reagent grade (MB, the Third Reagent Factory of Shanghai). Its stock solution of 1.0 /10 4 mol/l was prepared by directly dissolving its crystal into water. b-CD (95%, Yunnan Gourmet Factory) was recrystallized twice from double-distilled water before use. HP-b-CD (average MW /1657), degree of substitution (D.S. /9.0); sodium salt of CM-b-CD was prepared based on our previous research [18], (D.S. /4.8). Phosphate buffer solution was used to control the pH-value. All other reagents were
3.1. Adsorption spectra of MB and determination of K value Fig. 1 clearly showed the absorption spectra of MB monomer (666 nm) and dimer (612 nm). The increase in the MB monomer peak (666 nm) and dimer (612 nm) were observed with increasing concentration of MB, whereas the absorbance ratio of MB at 666 and 612 nm was obviously decreased with increasing concentration of MB (in Fig. 2). This indicated the absorption spectra of the MB were highly dependent on the concentration of the MB. It was caused by the existence of the monomer/dimer equilibrium of MB. 2MXD
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concentration of added MB.om and od represent the apparent extinction coeffcient of monomer and dimer, respectively. A is the absorbance of the solution. The dimerization constant K was calculated from equation [19] and K value was K / (6.29/1.1) /104 l/mol. 3.2. Inclusion complexation of MB with CDs
Fig. 1. The absorption spectra of 6 /10 6, 8 /10 6, 1.2 / 10 5, 1.4 /10 5, 1.6 /10 5, 2.0 /10 5, 2.4 /10 5 and 3.2 /10 5 (mol/l) MB solution (from bottom to top) at pH 7.2.
MB itself existed absorption at 612 and 666 nm, respectively. In the presence of CM-b-CD, the original absorption maximum of MB at 612 and 666 nm slightly shifted to a longer wavelength by 5 and 10 nm, respectively (seen in Fig. 3). A large increase absorption in MB monomer peak and a slight decrease in MB dimer peak were also observed with increasing concentration of CM-bCD. An isosbestic point of the MB was observed which implies there existed monomer/dimer equilibrium at 620 nm. This clearly indicated suppression of the dimer formation by inclusion of the MB monomer in the CM-b-CD cavity. CDs precluded to some extent MB /MB interaction and break the aggregate of the MB. Thus, CDs
Fig. 2. A plot of absorption ratio of MB at 666 and 612 nm vs. the concentration of MB.
A ct o m 0:25c2t [8Ko 2d =(o d o m )] A2 [2K=(o d o m )] Act [ 4Ko d =(o d o m )] [19] where K represents the equilibrium constant for the monomer(M)/dimer(D) equilibrium. ct is the
Fig. 3. The absorption spectra of MB (1 /10 5 mol/l) in the presence of different concentration of CM-b-CD at pH 7.2. Concentration of CM-b-CD (mol/l); 1, 0.000; 2, 2 /10 4; 3, 4 /10 4; 4, 6 /10 4; 5, 8 /10 4; 6, 1.6 /10 3; 7, 2 /10 3; 8, 3 /10 3.
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increased the fraction of the MB monomer by the formation of MB-CDs. Similar spectral changes in the MB solution were also observed upon addition of b-CD and HP-b-CD.
3.3. Fluorescence spectra of MB in CDs solution Fig. 4 showed that MB itself could only emit relatively weak fluorescence spectra in the absence of CDs. The maximum wavelengths of excitation and emission were at 664 and 686 nm, respectively. Addition of different CDs (b-CD, HP-b-CD, CMb-CD), an enhanced fluorescence emission of MB was observed. Maximum emission wavelength shifted to varying degree towards longer wavelength. Whereas the maximum excitation wavelength slightly shifted to shorter wavelength. These changes were due to the interaction between MB and CDs, implying the formation of MB-CDs inclusion complexes. The increase of fluorescence intensity resulted from the increase of fluorescence quantum yield, which came from the increase of electronic density after MB molecule entered the hydrophobic cavity of CDs. It was the hydrophobic cavity of CDs that provided the microenvironment of the high electronic density for MB molecule. Comparing absorption and excitation spectra of the MB, the excitation spectrum exhibited little response at the wavelength region of dimer absorption (l /612 nm). This suggested strongly that the monomer of the MB was
Fig. 4. Fluorescence properties of MB in different media; (1) H2O, (2) b-CD, (3) HP-b-CD, (4) CM-b-CD.
fluorescent while the dimer was virtually nonfluorescent. The nonfluorescent nature of the MB dimer was probably due to self-quenching of dimer [20].
3.4. Effect of CDs concentration MB concentration was held constant at 1.0 / 10 5 mol/l.While CDs were varied from 3.0 / 10 4 to 5.0 /10 3 mol/l. Fig. 5 showed the influence of the concentration of CDs on the fluorescence intensity of the MB. The fluorescence intensity of the MB was gradually enhanced with an increase of CDs (b-CD or HP-b-CD or CM-bCD) concentration until the stable inclusion complex was formed. It was noted that the most effective host molecule for MB was CM-b-CD.
3.5. Influence of pH MB and CDs concentrations were held constant 1.0 /10 5 and 1.0 /10 3 mol/l, respectively. The pH was changed from 2.0 to 11.0. Fig. 6 showed the influence of pH on the fluorescence intensities of the MB-CDs complex. Fluorescence intensities of MB-b-CD and MB-HP-b-CD were not basically varied at different pH values. While, fluorescence intensity of MB-CM-b-CD complex was weaker in acidic media than in neutral or basic media.
Fig. 5. Dependence of fluorescence intensities of MB on CD concentrations; (") b-CD, (') HP-b-CD, (m) CM-b-CD.
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CM-b-CD/MB-HP-b-CD /MB-b-CD. Because their cavities become larger and flexible than parent CD, the chemically modified CDs can alter their complexing capacity. Formation constants of MB at different pH values in the different CD systems were shown in Fig. 7. The MB-CM-b-CD formation constants was more sensitive to pH values than MB-b-CD and MB-HP-b-CD. This implied that the selective inclusion process associated with the species form of MB and CDs. Fig. 6. Dependence of fluorescence intensities of MB-CDs on pH; (") b-CD, (') HP-b-CD, (m) CM-b-CD.
3.6. Formation constants of MB-CD complexes Inclusion formation constant (K ) was a measure for complexing capacity of CDs. The formation constants of MB with CD (b-CD or HP-b-CD or CM-b-CD) were evaluated at different pH values assuming a 1:1 (CD:MB) inclusion model. The inclusion process is as follows: CDMBX CD MB where the symbols CD, MB, and CD-MB represent cyclodextrin (b-CD or HP-b-CD or CM-bCD), methylene blue, and the inclusion complex, respectively. The formation constant can be obtained from fluorescence data by the modified Benesi /Hildebrand equation (double be reciprocal plot) [21]. 1=(F F0 ) 1=fKkQ[P]0 [CD]0 g1=(kQ[P]0 ) where F and F0 represent the fluorescence signals of MB in the presence and absence of CD; [P ]0 and [CD ]0 represent the initial concentration of MB and CD; k is an instrumental constant; K is the formation constant of the complex; Q is the quantum yield for the complex. K can be obtained from the slope and intercept. Table 1 listed the linear-regression equations 1/[F /F0] versus 1/ [CD ]0 for MB to CDs at pH 7.2. The plots exhibited good linearity. This implied that the formation of inclusion complexes with a stoichiometry of 1:1 (MB:b-CD or MB:HP-b-CD or MB:CM-b-CD). The formation constants of the MB-CD complexes were also listed in Table 1. The formation constants (K ) followed the order: MB-
3.7. The related inclusion mechanism It is generally believed that dipole /dipole, electrostatic, van der Walls forces, hydrogen bonding, hydrophobic interaction, and the release of distortion energy of CD ring upon guest binding cooperatively govern the stability of an inclusion complex [22,23]. The hydrophobic substituents introduced at the rim of a CD cavity upon guest accommodation, and the molecular recognition by chromophoric CD is achieved through the induced-fit mechanism. MB is an ionic species with positive electric charge.
The neutral CDs are not charged (2 B/pH B/11) and the major inclusion interactions are hydrophobic interactions between the guest and CD cavity and hydrogen bonding of the guest to /OH groups or other introduced groups on CD ring. The experimental results showed that CDs (b-CD, HP-b-CD and CM-b-CD) easily includes MB in neutral and basic media. Moreover, we noted that CM-b-CD exhibited a stronger binding capacity than b-CD and HP-b-CD under the experimental conditions. This is because all carboxylic groups are deprotonated for CM-b-CD (pKa B/4) at pH / 5. Introduced charges on the CD are negatively charged. However, MB is positively charged. Thus, additional electrostatic interaction between negatively charged CM–CD and positively
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Table 1 Formation constants of MB-CD in different CDs at pH 7.2 Media
K R The linear-regression equations a
b-CD
HP-b-CD
CM-b-CD
4609/15 (621a) 0.998 y /1 /10 5x/0.0044
6559/25 0.990 y /1 /10 5x/0.0067
9289/47 0.993 y /1 /10 6x/0.0044
[16].
properties of MB. Therefore, there will be potential applications in mimetic enzyme and biological probe.
Acknowledgements
Fig. 7. Formation constants for MB-CDs at different pH; pH 2, pH 7.2, pH 11.
charged MB leads to stronger binding properties. In acidic media (pH 2) that CM-b-CD has a weaker binding capacity than b-CD and HP-bCD. This is due to the protonation of carboxylic groups in b-CD molecular. So, the formation constants of MB with CM-b-CD is very more sensitive to the changes of pH values than HP-bCD and b-CD.
4. Conclusion Neutral and charged CDs breaking the aggregate of the MB and forming 1:1 inclusion complexes have been studied by absorption and fluorescence spectroscopy. Especially, the charged CM–CD exhibits additional electrostatic inclusion characteristics, which is different from the neutral CD and its derivates. Its conclusion capacity also depends on the molecular species in aqueous solution except for the size-match and hydrophobicity. CDs include MB to form host /guest complex and alter the physical and chemical
This work was supported by the National Natural Science Foundation of China (Number 20172035) and Shanxi Province. Meanwhile, it was also supported by the National Education Committee Foundation for Outstanding Young Teachers.
References [1] P.V. Kamat, N.N. Lichtin, J. Phys. Chem. 85 (1981) 814. [2] J. Ye, P.R. Baldwin, Anal. Chem. 60 (1988) 2263. [3] M. Ohtani, S. Kuwabata, H.J. Yoneyama, Electroanal. Chem. 422 (1997) 45. [4] S. Braslavaky, T. Gensch, J. Phys. Chem. B 101 (1997) 101. [5] G.J.S. Foncer, R.C. Reej, R. Devonshire, Photochem. Photobiol. 52 (1990) 489. [6] E. Tuite, B. Norden, J. Am. Chem. Soc. 116 (1994) 7548. [7] R. Elena, J. Giulo, Rev. Chem. Intermediates 10 (1998) 260. [8] M.L. Bender, M. Komiyama, Cyclodextrin Chemistry, Springer, Berlin, 1978. [9] W. Saenger, Angew. Chem., Int. Ed. Engl. 19 (1980) 334. [10] G. Puglisi, N.A. Santagati, R. Pignatello, Durg Dev. Ind. Pharm. 16 (1990) 395. [11] M. Matsushima, Japan Patent 94 1994 343, 419. [12] Microgenic Corp., EU Patent 301 1989 847. [13] F.G. Sanchez, M.H. Lopez, J.C.M. Gomez, Analyst 112 (1987) 1037. [14] G. Zhao, J. Zhu, J. Zhang, Anal. Chim. Acta 394 (1999) 337 /344. [15] T. Matsue, D.H. Evans, T. Osa, N. Kobayashi, J. Am. Chem. Soc. 107 (1985) 3411.
G. Zhang et al. / Spectrochimica Acta Part A 59 (2003) 2935 /2941 [16] Z. Yuan, M. Zhu, S. Han, Anal. Chim. Acta 389 (1999) 291 /298. [17] G. Zhao, J. Zhu, H. Chen, Spectrochim. Acta Part A 55 (1999) 1109 /1117. [18] S. Shaomin, M.F. Choi, J. Chromatogr., A 919 (2) (2001) 321 /329. [19] X. Zhang, J. Ma, P. Xia, Sci. Chin. Ser. B 3 (1992) 225.
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[20] S. Jockusch, D. Lee, N.J. Turro, E.F. Leonard, Proc. Natl. Acad. Sci. USA 93 (1996) 7446. [21] G.C. Catena, F.V. Bright, Anal. Chem. 61 (1989) 905. [22] Y. Inoue, T. Hakuhsi, Y. Liu, L.-H. Tong, B.-J. Shen, D.S. Jin, J. Am. Chem. Soc. 115 (1993) 475. [23] Y. Inoue, Y. Liu, L.-H. Tong, B.-J. Shen, D.-S. Jin, J. Am. Chem. Soc. 115 (1993) 10637.
Study on the interaction of methylene blue with cyclodextrin derivatives by absorption and fluorescence spectroscopy Guomei Zhang, Shaomin Shuang *, Chuan Dong, Jinghao Pan Department of Chemistry, Shanxi University, Shanxi, Taiyuan 030006, China Received 18 December 2002; accepted 5 February 2003
Abstract The ability of b-cyclodextrin (b-CD), hydroxypropyl-b-cyclodextrin (HP-b-CD), and carboxymethyl-b-cyclodextrin (CM-b-CD) to break the aggregate of the methylene blue (MB) and to form 1:1 inclusion complexes has been studied by absorption and fluorescence spectroscopy. Experimental conditions including concentrations of various cyclodextrins (b-CD, HP-b-CD and CM-b-CD) and media acidity were investigated for the inclusion formation in detail. The formation constants are calculated by using steady-state fluorimetry, from which the inclusion capacity of different cyclodextrins (CDs) is compared. The results suggest that the charged b-cyclodextrin (CM-b-CD) is more suitable for inclusion of the cationic dye MB than the neutral b-cyclodextrins (b-CD, HP-b-CD) at pH/5. A mechanism is proposed which is consistent with the stronger binding of MB with CM-b-CD compared with the other CDs at pH/5. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Cyclodextrin; Methylene blue; Fluorescence
1. Introduction A wide variety of application of the phenothiazine dyes have been reported, for example, as sensitizers in solar energy conversion [1], redox mediators in catalytic oxidation reactions [2], active species in electrochromism [3] and dye lasers [4], ingredients in pharmaceutical preparation [5], and candidates for cancer therapy by intercalating between DNA base layers [6]. However, all of these applications are often complicated due to the
* Corresponding author. Fax: /86-351-701-1322. E-mail address: [email protected] (S. Shuang).
dimerization of dye molecules in aqueous media such as the great decrease of light sensitivity [7], which results from increased inner conversion of dimer exciter state. Thus, it is an important clue to improve the sensitivity of dyes towards obtaining a great number of monomers. Cyclodextrins (CDs) is one of the most important host molecules in supramolecular chemistry. CDs have the peculiar ‘interior hydrophobic, exterior hydrophilic’ structure forming a 1:1 or 1:2 inclusion complex with gust molecules, thus the physical, chemical and biochemical characters of guest molecules are modified [8,9]. The property of CDs can be used to control the position of monomer/dimer equilibrium and aggregation be-
1386-1425/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1386-1425(03)00123-9
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havior of dye, affecting electronic absorption and fluorescence spectra and photochemical and electrochemical properties of dyes. Different inclusion models have been observed, e.g. inclusion within the cavity or binding to the rim or penetrating partially into the CDs. CDs with inclusion properties have been widely used in pharmaceutical [10], food [11], environmental protection analysis [12], and enzyme modeling [13]. Methylene blue (MB) is one of phenothiazine dyes with a planar structure. It is today a generally accepted concept that planar dye molecules, such as acridine dyes, bind to DNA ‘intercalation’ mode. The interaction of planar molecules with DNA focuses on the aspects of biological probes and in the design of the efficient drugs targeted to DNA. Many analytical methods have been reported for the investigation on the complex of CD with MB including electrochemical method [14,15] and UV /Vis spectrotometry [16,17], etc. The recent methods are limited to parent b-CD. The purpose of this paper is to study the complexing behavior of MB with charged CD derivative CM-b-CD and neutral b-CD and HP-bCD by absorption and stead-state fluorescence spectroscopy technique. The complexing capacity of CDs with MB was compared. The possible inclusion mechanism was proposed that the charged CD was different from that of the neutral CDs.
analytical-reagent without purification. Double distilled water was used throughout. 2.2. Apparatus The absorption and fluorescence measurements were performed with a UV-265 spectrophotometer (Shimadzu, Japan), and a F-4500 spectrofluorimeter (Hitachi, Japan), respectively. Excitation and emission bandwidths were both set at 5 nm. pHs-2 meter was made in the 2nd Instrument Factory of Shanghai in China. All experiments were carried out at 209/1 8C. 2.3. Procedures A 1 ml aliquot of the stock solution (1.0 /10 4 mol/l) of MB was transferred into a 10 ml volumetric flask, then appropriate amount of 0.01 mol/l b-CD (or HP-b-CD or CM-b-CD) solution was added. The pH was controlled by 0.5 mol/l phosphate buffer solution. The mixed solution was diluted to final volume with distilled water and shaken thoroughly, following equilibrated for 30 min at 20 8C. All the measurements of absorption, fluorescence were made against the blank solution treated in the same way without CDs by using 1.0 cm quartz cell.
3. Results and discussion 2. Experimental 2.1. Reagents MB was of analytical reagent grade (MB, the Third Reagent Factory of Shanghai). Its stock solution of 1.0 /10 4 mol/l was prepared by directly dissolving its crystal into water. b-CD (95%, Yunnan Gourmet Factory) was recrystallized twice from double-distilled water before use. HP-b-CD (average MW /1657), degree of substitution (D.S. /9.0); sodium salt of CM-b-CD was prepared based on our previous research [18], (D.S. /4.8). Phosphate buffer solution was used to control the pH-value. All other reagents were
3.1. Adsorption spectra of MB and determination of K value Fig. 1 clearly showed the absorption spectra of MB monomer (666 nm) and dimer (612 nm). The increase in the MB monomer peak (666 nm) and dimer (612 nm) were observed with increasing concentration of MB, whereas the absorbance ratio of MB at 666 and 612 nm was obviously decreased with increasing concentration of MB (in Fig. 2). This indicated the absorption spectra of the MB were highly dependent on the concentration of the MB. It was caused by the existence of the monomer/dimer equilibrium of MB. 2MXD
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concentration of added MB.om and od represent the apparent extinction coeffcient of monomer and dimer, respectively. A is the absorbance of the solution. The dimerization constant K was calculated from equation [19] and K value was K / (6.29/1.1) /104 l/mol. 3.2. Inclusion complexation of MB with CDs
Fig. 1. The absorption spectra of 6 /10 6, 8 /10 6, 1.2 / 10 5, 1.4 /10 5, 1.6 /10 5, 2.0 /10 5, 2.4 /10 5 and 3.2 /10 5 (mol/l) MB solution (from bottom to top) at pH 7.2.
MB itself existed absorption at 612 and 666 nm, respectively. In the presence of CM-b-CD, the original absorption maximum of MB at 612 and 666 nm slightly shifted to a longer wavelength by 5 and 10 nm, respectively (seen in Fig. 3). A large increase absorption in MB monomer peak and a slight decrease in MB dimer peak were also observed with increasing concentration of CM-bCD. An isosbestic point of the MB was observed which implies there existed monomer/dimer equilibrium at 620 nm. This clearly indicated suppression of the dimer formation by inclusion of the MB monomer in the CM-b-CD cavity. CDs precluded to some extent MB /MB interaction and break the aggregate of the MB. Thus, CDs
Fig. 2. A plot of absorption ratio of MB at 666 and 612 nm vs. the concentration of MB.
A ct o m 0:25c2t [8Ko 2d =(o d o m )] A2 [2K=(o d o m )] Act [ 4Ko d =(o d o m )] [19] where K represents the equilibrium constant for the monomer(M)/dimer(D) equilibrium. ct is the
Fig. 3. The absorption spectra of MB (1 /10 5 mol/l) in the presence of different concentration of CM-b-CD at pH 7.2. Concentration of CM-b-CD (mol/l); 1, 0.000; 2, 2 /10 4; 3, 4 /10 4; 4, 6 /10 4; 5, 8 /10 4; 6, 1.6 /10 3; 7, 2 /10 3; 8, 3 /10 3.
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increased the fraction of the MB monomer by the formation of MB-CDs. Similar spectral changes in the MB solution were also observed upon addition of b-CD and HP-b-CD.
3.3. Fluorescence spectra of MB in CDs solution Fig. 4 showed that MB itself could only emit relatively weak fluorescence spectra in the absence of CDs. The maximum wavelengths of excitation and emission were at 664 and 686 nm, respectively. Addition of different CDs (b-CD, HP-b-CD, CMb-CD), an enhanced fluorescence emission of MB was observed. Maximum emission wavelength shifted to varying degree towards longer wavelength. Whereas the maximum excitation wavelength slightly shifted to shorter wavelength. These changes were due to the interaction between MB and CDs, implying the formation of MB-CDs inclusion complexes. The increase of fluorescence intensity resulted from the increase of fluorescence quantum yield, which came from the increase of electronic density after MB molecule entered the hydrophobic cavity of CDs. It was the hydrophobic cavity of CDs that provided the microenvironment of the high electronic density for MB molecule. Comparing absorption and excitation spectra of the MB, the excitation spectrum exhibited little response at the wavelength region of dimer absorption (l /612 nm). This suggested strongly that the monomer of the MB was
Fig. 4. Fluorescence properties of MB in different media; (1) H2O, (2) b-CD, (3) HP-b-CD, (4) CM-b-CD.
fluorescent while the dimer was virtually nonfluorescent. The nonfluorescent nature of the MB dimer was probably due to self-quenching of dimer [20].
3.4. Effect of CDs concentration MB concentration was held constant at 1.0 / 10 5 mol/l.While CDs were varied from 3.0 / 10 4 to 5.0 /10 3 mol/l. Fig. 5 showed the influence of the concentration of CDs on the fluorescence intensity of the MB. The fluorescence intensity of the MB was gradually enhanced with an increase of CDs (b-CD or HP-b-CD or CM-bCD) concentration until the stable inclusion complex was formed. It was noted that the most effective host molecule for MB was CM-b-CD.
3.5. Influence of pH MB and CDs concentrations were held constant 1.0 /10 5 and 1.0 /10 3 mol/l, respectively. The pH was changed from 2.0 to 11.0. Fig. 6 showed the influence of pH on the fluorescence intensities of the MB-CDs complex. Fluorescence intensities of MB-b-CD and MB-HP-b-CD were not basically varied at different pH values. While, fluorescence intensity of MB-CM-b-CD complex was weaker in acidic media than in neutral or basic media.
Fig. 5. Dependence of fluorescence intensities of MB on CD concentrations; (") b-CD, (') HP-b-CD, (m) CM-b-CD.
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CM-b-CD/MB-HP-b-CD /MB-b-CD. Because their cavities become larger and flexible than parent CD, the chemically modified CDs can alter their complexing capacity. Formation constants of MB at different pH values in the different CD systems were shown in Fig. 7. The MB-CM-b-CD formation constants was more sensitive to pH values than MB-b-CD and MB-HP-b-CD. This implied that the selective inclusion process associated with the species form of MB and CDs. Fig. 6. Dependence of fluorescence intensities of MB-CDs on pH; (") b-CD, (') HP-b-CD, (m) CM-b-CD.
3.6. Formation constants of MB-CD complexes Inclusion formation constant (K ) was a measure for complexing capacity of CDs. The formation constants of MB with CD (b-CD or HP-b-CD or CM-b-CD) were evaluated at different pH values assuming a 1:1 (CD:MB) inclusion model. The inclusion process is as follows: CDMBX CD MB where the symbols CD, MB, and CD-MB represent cyclodextrin (b-CD or HP-b-CD or CM-bCD), methylene blue, and the inclusion complex, respectively. The formation constant can be obtained from fluorescence data by the modified Benesi /Hildebrand equation (double be reciprocal plot) [21]. 1=(F F0 ) 1=fKkQ[P]0 [CD]0 g1=(kQ[P]0 ) where F and F0 represent the fluorescence signals of MB in the presence and absence of CD; [P ]0 and [CD ]0 represent the initial concentration of MB and CD; k is an instrumental constant; K is the formation constant of the complex; Q is the quantum yield for the complex. K can be obtained from the slope and intercept. Table 1 listed the linear-regression equations 1/[F /F0] versus 1/ [CD ]0 for MB to CDs at pH 7.2. The plots exhibited good linearity. This implied that the formation of inclusion complexes with a stoichiometry of 1:1 (MB:b-CD or MB:HP-b-CD or MB:CM-b-CD). The formation constants of the MB-CD complexes were also listed in Table 1. The formation constants (K ) followed the order: MB-
3.7. The related inclusion mechanism It is generally believed that dipole /dipole, electrostatic, van der Walls forces, hydrogen bonding, hydrophobic interaction, and the release of distortion energy of CD ring upon guest binding cooperatively govern the stability of an inclusion complex [22,23]. The hydrophobic substituents introduced at the rim of a CD cavity upon guest accommodation, and the molecular recognition by chromophoric CD is achieved through the induced-fit mechanism. MB is an ionic species with positive electric charge.
The neutral CDs are not charged (2 B/pH B/11) and the major inclusion interactions are hydrophobic interactions between the guest and CD cavity and hydrogen bonding of the guest to /OH groups or other introduced groups on CD ring. The experimental results showed that CDs (b-CD, HP-b-CD and CM-b-CD) easily includes MB in neutral and basic media. Moreover, we noted that CM-b-CD exhibited a stronger binding capacity than b-CD and HP-b-CD under the experimental conditions. This is because all carboxylic groups are deprotonated for CM-b-CD (pKa B/4) at pH / 5. Introduced charges on the CD are negatively charged. However, MB is positively charged. Thus, additional electrostatic interaction between negatively charged CM–CD and positively
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Table 1 Formation constants of MB-CD in different CDs at pH 7.2 Media
K R The linear-regression equations a
b-CD
HP-b-CD
CM-b-CD
4609/15 (621a) 0.998 y /1 /10 5x/0.0044
6559/25 0.990 y /1 /10 5x/0.0067
9289/47 0.993 y /1 /10 6x/0.0044
[16].
properties of MB. Therefore, there will be potential applications in mimetic enzyme and biological probe.
Acknowledgements
Fig. 7. Formation constants for MB-CDs at different pH; pH 2, pH 7.2, pH 11.
charged MB leads to stronger binding properties. In acidic media (pH 2) that CM-b-CD has a weaker binding capacity than b-CD and HP-bCD. This is due to the protonation of carboxylic groups in b-CD molecular. So, the formation constants of MB with CM-b-CD is very more sensitive to the changes of pH values than HP-bCD and b-CD.
4. Conclusion Neutral and charged CDs breaking the aggregate of the MB and forming 1:1 inclusion complexes have been studied by absorption and fluorescence spectroscopy. Especially, the charged CM–CD exhibits additional electrostatic inclusion characteristics, which is different from the neutral CD and its derivates. Its conclusion capacity also depends on the molecular species in aqueous solution except for the size-match and hydrophobicity. CDs include MB to form host /guest complex and alter the physical and chemical
This work was supported by the National Natural Science Foundation of China (Number 20172035) and Shanxi Province. Meanwhile, it was also supported by the National Education Committee Foundation for Outstanding Young Teachers.
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