In the Laboratory
W
Cyclodextrin Inclusion Complexes with a Solvatochromic Fluorescent Probe Nicole J. Crane, Rudolph C. Mayrhofer, and Thomas A. Betts* Department of Physical Sciences, Kutztown University of Pennsylvania, Kutztown, PA 19530; *
[email protected] Gary A. Baker Department of Chemistry, State University of New York, Buffalo, NY 14260
PRODAN. Students have the opportunity to use fluorescence spectroscopy to explore solvatochromism, determine formation constants, and employ mathematical modeling. Variations to the procedures described here will allow students to investigate the effects of pH, temperature, ionic strength, and derivatized CDs on complex formation. This laboratory experiment would likely be used in the chemistry curriculum after students have executed a quantitative determination using fluorescence, and have some familiarity with fluorescence theory and instrumentation. O
–
CH2CH3
S1 H3C
+
N
H3C
hν
O
Flb
Flr CH2CH3
.. H3C
S0
N
H3C
Figure 1. Simplified Jablonski diagram indicating solvent relaxation, and electron shuffling (intramolecular charge transfer) for PRODAN upon excitation from the ground (S0) to the first excited electronic state (S1). Flb represents the blueshifted fluorescence from the unrelaxed state, and Flr represents the redshifted fluorescence from the relaxed state.
1.2
Normalized Fluorescence Intensity
Solvent polarity can have a dramatic effect on the emission color of certain fluorescent probes. Such solventsensitive—solvatochromic—probes have as a defining feature the simultaneous presence of electron-donating and electronwithdrawing groups in resonant positions in a conjugated system. When a solvatochromic molecule absorbs a photon, the dipole moment increases as a result of charge traveling along the system, intramolecular charge transfer (see Figure 1). The energy of this excited state, S1, which is no longer in equilibrium with the surrounding solvation shell, can be lowered, or relaxed, by the reorganization of polar solvent molecules which create favorable interactions with the increased dipole of the fluorescent molecule. The underpinnings of the solvent-induced spectral shift rely on this solvent reorganization known as solvent relaxation. If solvent relaxation occurs prior to the radiative transition to the ground state, S0, the result is a lower energy excited state and a concomitant red shift in the fluorescence emission (Flr). In less-polar solvents this relaxation process is less effective resulting in blueshifted emission (Flb) relative to that in polar media. The classic example of such a donor–acceptor molecule exhibiting solvatochromism is 6-propionyl-2-(N,Ndimethylamino)naphthalene, PRODAN (1), whose groundand excited-state dipole moments are 2.8 D and 10 D (2), respectively. The emission maximum of PRODAN shifts 150 nm between solutions of cyclohexane and water (Figure 2). The tremendous sensitivity of PRODAN emission to solvent polarity has been used to investigate the binding sites of proteins (1, 3, 4) and enzymes (5), interactions with cell membranes and phospholipid vesicles (6–8), the structure of reverse micelles (9, 10), and cyclodextrin inclusion complexation (11). Cyclodextrins, CDs, are cyclic oligosaccharides that have a hydrophobic cavity with a hydrophilic exterior due to the primary and secondary hydroxyl groups positioned at the rims. CDs composed of six, seven, or eight glucopyranose units are known as α-, β-, and γ-CD, respectively. The inclusion of guest molecules in the CD cavity is driven by the hydrophobic effect, van der Waals interactions, and displacement of water from the hydrophobic cavity, as well as cavity size and shape considerations. CD research based on this complexation has exploded over the past decade. Applications in odor control (12) and drug delivery (13, 14) have been cited, but CDs have been used extensively in chromatography (15, 16) and capillary electrophoresis (15, 17). Several articles have appeared in this Journal describing experiments involving CD inclusion complexes (18–20). In this laboratory experiment the environment-sensitive nature of PRODAN fluorescence is used to determine formation constants for the complexation of β- or γ-CD with
1.0
chloroform ethanol cyclohexane DMSO
water
0.8 0.6 0.4 0.2 0.0 350
400
450
500
550
600
Emission Wavelength / nm Figure 2. Normalized fluorescence emission spectra of 1µM PRODAN in cyclohexane, chloroform, dimethylsulfoxide (DMSO), ethanol, and water (λex = 360 nm).
JChemEd.chem.wisc.edu • Vol. 79 No. 10 October 2002 • Journal of Chemical Education
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In the Laboratory
Experimental
Hazards The hazards of PRODAN have not been thoroughly investigated; therefore this compound should be handled as potentially hazardous. Chloroform is a suspected cancer hazard. Cyclohexane, chloroform, dimethyl sulfoxide, methanol, and ethanol are flammable. Dimethyl sulfoxide is quickly absorbed through the skin and is capable of transporting other dissolved substances into the body. Cyclodextrin dust is flammable and explosive. Results and Discussion The effect of increasing β- and γ-CD concentration on PRODAN emission spectra is shown in Figure 3. For β-CD (Panel A) the emission blueshifts slightly and the fluorescence intensity increases as the concentration of β-CD increases. As the concentration of γ-CD increases (Panel B), a fluorescent component near 430 nm becomes more intense. This dramatic blueshift in the emission profile indicates that PRODAN is experiencing an environment that is much less dipolar than water. A comparison of this emission maximum to the spectra in Figure 2 provides an estimate of the polarity of the environment in which PRODAN resides. The blueshifts in PRODAN emission along with the increased intensities in these two systems are due to inclusion of PRODAN in the hydrophobic cavities of the CDs. 1262
0.0135 M β-CD
12
A
10 8 6
Fluorescence Intensity / 10 5
PRODAN was purchased from Molecular Probes (Eugene, OR); β- and γ-CD were purchased from Cerestar, USA (Hammond, IN), and ethanol was purchased from McCormick Distilling Co. (Weston, MS). In order to establish the polarity-sensitive nature of PRODAN, students were directed to collect emission spectra for 1 µM PRODAN dissolved in solvents of varying polarity (e.g., cyclohexane, chloroform, dimethylsulfoxide, methanol or ethanol, and water). All solvents except water were dried over molecular sieves. An aliquot of ethanolic PRODAN stock solution was added to a quartz cuvette, and the ethanol was removed by a gentle nitrogen stream before the dried solvent of interest was added. In order to highlight the spectral shift we recommend that students normalize these fluorescence emission spectra (Figure 2). For the determination of the PRODAN/γ-CD formation constant a series of eight aqueous solutions were prepared in disposable methacrylate cuvettes (A. Daigger & Co.) in which the PRODAN concentration was identical (1 µM) and γ-CD concentration was varied from 0 to 0.10 M. For the PRODAN/β-CD system, the β-CD concentration of eight aqueous 1 µM PRODAN solutions was varied from 0 to 0.0135 M. After thorough mixing, emission spectra (λem = 400–600 nm) were recorded for all solutions (λex = 375 nm). A Photon Technologies International C-61 fluorescence spectrometer was used to collect all emission spectra. The laboratory experiment makes use of a fluorescence spectrophotometer capable of collecting excitation and emission spectra; however, we have also used a Turner Model 112 filter fluorometer to determine equilibrium constants for the systems.
4
H2O
2 0 14 12
0.100 M γ-CD
B
10 8 6 4 2 0 400
H2O 450
500
550
600
650
Emission Wavelength / nm Figure 3. Fluorescence emission spectra of PRODAN in varying concentrations of CDs. Panel A: PRODAN in β-CD solutions. In order of increasing intensity; [β-CD] = 0, 0.0003, 0.0006, 0.001, 0.004, 0.007, 0.010, 0.0135 M (λ ex = 385 nm). Panel B: PRODAN in γ-CD solutions. In order of increasing intensity at 430 nm; [γ-CD] = 0, 0.007, 0.010, 0.015, 0.025, 0.050, 0.070, 0.100 M (λex = 375 nm).
The blueshift is not nearly as dramatic for the PRODAN/ β-CD system as it is for PRODAN/γ-CD. First, the cavity of β-CD is smaller than that of γ-CD, and may allow a portion of PRODAN to remain exposed to the solvent. Thus, the emission maximum of PRODAN in β-CD would reflect a polarity intermediate between that of the solvent and the cavity. The cavity of γ-CD has a larger diameter, and could more effectively shield PRODAN from the solvent. Another possible interpretation is a higher order complex (i.e., 2:1 or 1:2 CD:PRODAN stoichiometry). Because the fluorescence intensity change is due to the degree of complexation of PRODAN in the CD cavity, it is possible to use a Benesi–Hildebrand approach to determine the formation constants of the complexes. The derivation of this method as it applies to CD complexation has been described elsewhere (21). Assuming the stoichiometry is 1:1, F⫺F0 versus [CD] should fit the following equation: 1 1 1 = + (1) ( F − F0 ) ( F∞ − F0 )K 1[CD] ( F∞ − F0 ) where F0 is the fluorescence intensity in the absence of CD, F is the fluorescence intensity at a particular concentration of CD, F⬁ is the fluorescence intensity when all of the PRODAN is complexed, and K1 is the equilibrium constant for the 1:1 complex. If two CDs complexed one PRODAN (stoichimetry 2:1), F–F0 versus [CD] should be well described by the following equation:
Journal of Chemical Education • Vol. 79 No. 10 October 2002 • JChemEd.chem.wisc.edu
In the Laboratory
(F – F0) / 10 5
This experiment can also provide an opportunity to investigate the effects of additional environmental parameters on complex formation. For example, if each student determined the equilibrium constant for one system at a different pH, the class could cooperatively determine the effect of pH on the formation of that complex. Similarly, classes of students could investigate the effects of temperature, ionic strength, addition of alcohols, and CD derivatization on complex formation.
A
8
6
4
2
W 0 0
3
6
9
12
Supplemental Material
15
[β-CD] / (10᎑3 mol/L)
Instructions for students and notes for the instructor are available in this issue of JCE Online.
16
Acknowledgments
(F – F0) / 10 5
B
Support for the development of this laboratory was provided by the National Science Foundation (DUE 98-51660) and the Faculty Professional Development Council of the Pennsylvania State System of Higher Education. We would also like to thank Frank V. Bright and Emily D. Niemeyer for their assistance in laying the foundation for this work.
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Literature Cited
0 0
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[γ-CD] / (10᎑2 mol/L) Figure 4. F⫺F0 vs [CD] for β-CD (Panel A) and γ-CD (Panel B). Mathematical fit to a 1:1 binding model (—) using eq 1, and a 2:1 binding model (- - -) using eq 2. Fluorescence intensities were determined at 500 nm for β-CD and 430 nm for γ-CD
1 1 1 = + ( F − F0 ) ( F∞ − F0 )K 1K 2[CD]2 ( F∞ − F0 )
(2)
Nonlinear least-squares fitting software (TableCurve, Jandel Scientific) was used to fit the experimental data to the prescribed equation in each case. F⬁ and K1 (or K1K2) were allowed to float. The fitting results for both 1:1 and 2:1 binding are presented in Panels A and B of Figure 4 for β- and γCD, respectively. For the PRODAN/β-CD system the 1:1 stoichiometric model resulted in K1 = 2000 M᎑1, r 2 = .996, and the 2:1 model resulted in K1K2 = 4.96 x 106 M᎑2, r 2 = .959. Clearly, the 1:1 model fit best. For the PRODAN/γ-CD system the 1:1 stoichiometric model resulted in K1 = 26 M᎑1, r 2 = .983, and the 2:1 model resulted in K1K2 = 2700 M᎑2, r 2 = .993. In this case, the 2:1 model fit best, but the distinction was less apparent. Alternatively, double reciprocal plots can be constructed to determine the stoichiometry and formation constants for the complexes. For a complex with 1:1 stoichiometry, according to eq 1, a plot of 1/(F⫺F0) versus 1/[CD] results in a straight line, and K1 is determined by dividing the intercept by the slope. Likewise, for a complex with 2:1 stoichiometry, according to eq 2, a plot of 1/(F⫺F0) versus 1/[CD]2 results in a straight line, and K1K2 is determined by dividing the intercept by the slope.
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