9566
J. Phys. Chem. 1993, 97, 9566-9572
Inclusion of Fluorescein and Halogenated Derivatives in cy-, 8-, and y-Cyclodextrins. A Steady-State and Picosecond Time-Resolved Study Lucia Flamigni Istituto FRAE-CNR, Via de’ Castagnoli, 1 , 401 26 Bologna, Italy Received: March 9, 1993; In Final Form: May 12, 1993
The inclusion of fluorescein (FL), erythrosin B (EB), and rose bengal (RB) in a-, 0-, y-cyclodextrins (a-, 0-, and y-CDs) was studied with several techniques. Induced circular dichroism (icd) absorption spectroscopy and steady-state and picosecond time resolved fluorescence spectroscopy were used to derive stoichiometry, association constants, and structural information on the inclusion complexes formed. A stoichiometry of 1:l was found for the range of concentration explored (xanthenes, 10-6-10-4 M; CD, 10-3-10-2 M). From icd measurements, FL is found to bind weakly with a- and y-CDs ( K A= 35 M-l) and associate preferably with 0-CD ( K A = 360 M-l). EB binds preferably to y-CD ( K A = 110 M-I) and more weakly with a-and 0-CDs ( K A = 20 M-l, K A = 40 M-l, respectively). RB binds only with y-CD with an association constant similar to that of EB ( K A = 100 M-*, respectively). Fluorescence enhancement and different luminescence lifetimes with respect to that of the free chromophore in water are found only upon association of CD with EB and RB, the halogenated dyes. The nonradiative rate constant of their emitting state is known, in fact, to increase with the hydrogen bonding ability of the media. There is indication from two distinct fluorescence lifetimes, paralleled by different signs of icd bands, that at least two different complexes are formed, depending on the dimensions of the cavity and the degree of halogenation of the dye. The contemporary presence of the two complexes in y-CD systems could explain some inconsistency in the association constants determined by the different methods. FL complexes do not show fluorescence enhancement (except at high concentration of the dye where CD inclusion disrupts the nonluminescent aggregates), and a distinct emission lifetime with respect to water is not observed, as expected on the basis of the invariance of FL fluorescence yield and lifetimes with the nature of the medium.
Introduction Cyclodextrins (CDs) are water soluble cyclic oligosaccharides which, with their torus shape and relatively apolar interior, can selectively incorporate molecules on the basis of size and polarity characteristics.1J The commonly available cyclodextrins are a-CD (cyclohexaamilose), 8-CD (cycloheptaamilose),and y-CD (cyclooctaamilose),characterized by increasing cavity diameters.l” The series of compounds examined, fluorescein (FL), erythrosin B (EB), and rose bengal (RB) dianions, have a similar structure but different dimensions due to increasing halogen substitution. Recently a contribution from this laboratory has reported on the photoreactivity of the halogenated fluorescein EB in micellar systems.4 The present research continues in the same line of clarifying photobehavior of xanthenes in microheterogeneous systems. The lasing and emission properties of the laser dye fluorescein were shown to improve when dissolved in waterlj3-CD systemsS compared to pure water. Similar properties were found for other xantheniclaser dyes.54 An association constant for the rhodamine B/@-CDcomplex, K A = 2900 M-1, was reported.6 This value is surprisingly different from a K A = 7 M-I derived for the fluorescein/,%CD complex from induced excited-state optical activity proper tie^.^ Halogenated xanthenes are widely used as singlet oxygen photosensitizers,photoinitiators of polymerization, active media in solar energy conversion, and sensitizers of photodynamic damage in biological materials8 Neckers reported on the effect of covalently linking RB to @-CDwith respect to the photooxidation efficiency in the presence of a quencher or a receptor includedin CD.9 Todatenostudy on thestabilityofthecomplexes and on the effect of the inclusion on photophysical and photochemical properties of halogenated fluorescein has been reported. Such a study is expected to be of interest for the practical application listed above in consideration of the catalytic activity and the ability to mimic enzyme-active sites of cyclodextrins.
A further major point of interest is the following. Halogenated fluorescein derivatives exhibit luminescence properties strongly dependent on the hydrogen donating ability of the medium;lOJ1 this makes these dyes extremely good probes to test microenvironmental properties.I2-l4 It has been reported in a number of papersls-I7that the polarity of the CD cavity is similar to that of ethanol. More recently Fleming and co-workers18measured the rate of excited-state proton transfer of the complexes of 1-aminopyrene and a-naphthol with a modified j3-CD. Because of their dimensions, these molecules were protruding in water. These authors, on the basis of the rates found, assigned to water, in proximity of the hydroxyl rim, a basicity similar to that of a water/ethanol mixture containing 80%alcohol. The modification of the properties was assigned to the perturbation brought about by the extensive network of OH groups of the CD rim. The present study is expected to contribute to this point. Induced circular dichroism absorption spectroscopyand steadystate and picosecond time resolved fluorescence spectroscopy have been used to derive information on stoichiometry, association constants, and structures of the complexes. Data on the fluorescence lifetime of the inclusion complexes formed by EB and RB can give detailed information on the microenvironment experienced by the probe molecules.
Experimental Section a-Cyclodextrin and j3-cyclodextrin from Serva, y-cyclodextrin from Fluka, and D(+)-glUCOSefrom Merck were used as received. Methanol fluorimetric grade (MeOH) and absolute ethanol for spectroscopy (EtOH) were from Carlo Erba. EB was purified as previously reported.4 RB high purity from Aldrich and fluorescein disodium salt (uranine) from Lambdachrome laser dyes were used as received. Water was purified by passage through a Millipore Milli-Q system. Aqueous solutions of known dye concentration were used to dissolve weighted amounts of CD. Successive dilution used the
QQ22-3654/93/2091-9566%04.0Q/Q 0 1993 American Chemical Society
The Journal of Physical Chemistry, Vol. 97, No. 38, 1993 9567
Inclusion of Fluoresceins in Cyclodextrins
CHART I
EB
T 9.5
T 1
\
0A
i
I
FL a-CD 0 -CD y-CD
startingdye solution as solvent. This procedure ensured a constant concentration of the dye in the absence and in the presence of the various CD concentrations. The pH of the FL samples was kept basic (pH 11) by addition of NaOH to the starting dye solutions. Glucoseconcentrationwas kept at 6,7,or 8 times theconcentration of reference CD. The concentrations used were 106-10-4 and 10-3-10-2 M for dye and CD, respectively. Absorption spectra were measured by a Perkin-Elmer Lambda 5 spectrophotometer. Emission spectra were obtained with a Spex-Fluorolog-2 fluorimeter. Induced circular dichroism ( i d ) spectra were measured by a Jasco 5 - 5 0 dichrograph. The picosecond time-resolved fluorescence spectrometer was based on a Nd:YAG laser, a spectrograph, and a streak camera. The cavity dumped, actively/passively mode locked Nd:YAG laser (Continuum PY62-10) with two amplification stages is able to deliver pulses up to an energy of 120 mJ/pulse (1064 nm) at 10 Hz with 35-ps full width at half-maximum (fwhm). The second harmonic (A = 532 nm, vertically polarized) with an energy of 0.5 mJ/pulse was used to excite the samples. A small fraction of the laser beam was split to trigger the streak camera via ap.i.n. diode head (HAMAMATSU C 1083);necessary time adjustments on the trigger signal were performed by a delay unit (HAMAMATSU C1097). The laser beam was delayed by an optical line to allow for the necessary 19-30-ns triggering delay of the streak; and then it was focused to an area of 0.1 cm2 on the sample. The light emitted was collected at a right angle to the excitation and passed a polarizer placed at the magic angle (54.7O) with respect to vertical, to remove the effects of molecular
d e d d
SA
6.5 0.5A
reorientation. Neutral density or cutoff filters were used when necessary. The transmitted light was then focused into the entrance slit of a spectrograph (HR 250 Jobin-Yvon) equipped with a 300 grooves/mm grating. The light, horizontallydispersed by the spectrograph, entered the slit (15 pm) of the streak camera (Hamamatsu C1587 equipped with the fast single sweep unit M 1952) which produced a streak image containing both spectral and temporal informations. Typical images were the average of 200 events. Acquisitionand processing of the data were performed via a cooled charge-coupled device (CCD) camera (Hamamatsu C3140) and related software running on a personal computer. The two dimensional streak-camera/CCD image contains 5 12 time points by 480 wavelength points (about 165 nm). The sweep of the system can vary in steps from 0.5 to 15.4 ps/channel. Time profiles were averaged over 50 wavelength channels (ca.20 nm). The analysis of the profiles was performed on 256 data points with standard iterative nonlinear procedure according to single exponentials or to a linear combination of exponentials. The goodness of the fit was judged on the basis of x2 and on the distribution of residuals along the time axis. Deconvolution with the excitation profile was effective only for lifetimes of the order of 100 ps or less. The overall time resolution of the system is estimated to be 20 ps, the triggering jitter being the limiting factor. Nanosecond fluorescencelifetimes were obtained with a time correlated single-photon counting apparatus. All measurements were at room temperature on air-saturated samples. The molecular models of the xanthenes were drawn and related
I
I
0
0 '
>
a
m
.
-2.51
bl
;; ':
bl
e8 0
5
C
h 0 300
.
I C
400
500
h,nm Figure 1. Induced circular dichroism spectra of FL solution: (a) [FL] = 1.5 X 10-4M, [a-CD] = 3.0 X le2 M, optical path 0.2 cm (-), optical path2cm(--);(b) [FL] = 1.5X lVM,[B-CD] = 1.4X 10-2M,optical M, optical path 0.2 cm; (c) [FL] = 1.5 X 10-4 M, [y-CD] = 2.1 X lt2 path 0.2 cm. -2.5
dimensions were calculated by the use of the molecular modeling system Alchemy II.19
Results Absorption Spectra. The absorption spectra of EB, RB, and FL(1 X 10-5to6X IO-sM)ina-CD(4X 10-2M),@-CD(1.4 X 10-2 M) and 7-CD (3 X 10-2 M) show very little change with respect to those in water. A slight red shift by 2-3 nm and a small decrease (5-8%) in the molar absorption coefficients over the entire absorption region are general phenomena observed upon addition of cyclodextrins to the dye solutions. A similar effect is detected in dye solutions added with D(+)-glucose (noncyclic saccharide); it is therefore assigned to interaction with saccharide molecules rather than to inclusion. These results do not seem to agree with those previously repbrted for FL where a "strong quenching" of absorption by 8-CD was observed.5 Since the only difference in the two experiments lies in the origin of 8-CD used, we can tentatively identify this fact as a possible source of disagreement in the data. Induced Circular Dichroism Spectra. Addition of CD to dye solutions induces in most cases optical activity in the achiral xanthenes, whileD(+)-glucosedoes not induceany optical activity. This is ascribed to xanthene/CD complex formation. RB in aand B-CDs did not show any induced circular dichroism. In all the other cases the signals were weak but especially in the UV range, noiseless enough to allow a quantitative analysis. Figures 1and 2 show the icd spectra of the dye/CD complexes. A BenesiHildebrand p1ot2Oof the icd signals is linear, indicating that the stoichiometry of the complex is 1:1 and allowing derivation of the equilibrium constants of the complex formation, KA. These are reported in Table I. The light transmitted by the sample in the visible band was, in general, very low, and only qualitative considerations could be drawn. In some complexes the splitting of this band in two by opposite signs was evidenced, as reported in Table I. FluorescenceSpectra. Fluorescence is a property of the excited state, and, in principle, it could not be used to derive ground-state association parameters. However, given theshort singlet lifetimes involved, we can safely assume that the deactivation is faster than the relocation of the molecule. This allows us to use this property to derive ground-state association parameters.
I
I
300
400
d 500
A,nm
Figure 2. Induced circular dichroism spcctra of EB and RB solutions: (a) [EB] = 9.5 X 10-5 M, [a-CD] = 3.0 X 10-1M,optical path 0.2 cm (-), optical path 4 cm (- -); (b) [EB] = 9.5 X M, [@-CD]= 1.4 X 10-2 M, optical path 0.2 cm (-), optical path 4 cm (- -); (c) [EB] = 9.5X 10-5 M, [Y-CD] = 2.1 X 1W2M, optical path 0.2 cm (-), optical path 2 cm (- -); (d) [RBI = 6.7 X le5M, [y-CD] 2.1 X le2M, optical path 0.2 cm (-), optical path 2 cm (- -).
TABLE I: Equilibrium Constant for the Association of & EB, and RB to a-,8-, and yCD, Obtained by icd Spectra' FL a 35 10 -+ B 360 40 ++
*
Y
EB RB
B Y a
B
35
10 20* 10 40 f 20 110*20
++ -+ -+ ++
*
Y 100 20 ++ The signs of the visible bands are also shown (see text).
Enhancing of fluorescence is detected in EB and RB upon complexation with CD, as shown in Figures 3 and 4 for the case of the systems EB/y-CD and EB/a-CD. FL shows only a very small (5%) enhancing of fluorescence upon CD addition. Glucose does not enhance the fluorescence of the dyes. A quantitative treatment of the fluorescence signal is complicated, with respect to icd, by the fact that both complexed and uncomplexed dyes emit. Optically thin samples were excited at the isosbestic point of complexed and uncomplexed species (as derived by the absorption spectra); in these conditions the following treatment21 can be applied. The integrated fluorescence emission (I) can be expressed in terms of molar fraction of uncomplexed (fi) and complexed (f') forms
I = C(@$l+ @c3j-2) (1) where c is a constant depending on dye concentration and other experimental conditions and Q is the emission quantum yield of complexed (*cD) and uncomplexed (@o) species. In the absence
Inclusion of Fluoresceins in Cyclodextrins
The Journal of Physical Chemistry, Vol. 97, No. 38, 1993 9569
TABLE II: Equilibrium Constants for the Association of RB and EB with CD, KA,Obtained from Fluorescence Spectra and Ratio of Emission Quantum Yields in CD-Complexed and Aaueous Dye @~o/@o* KA Of-')
EB
a
B Y
@CD/@O
10 20 50
1.8 2.1 2.7
40
4.0
RB
H
B Y 0
0
550
600
TABLE IIk Fluorescence Lifetimes (ps) of FL, EB, and RB in Neat Solvents, CD Systems, and Glucose Solutions FLa EB RB
650
h,nm Figure 3. Emission spectra of [EB] = 5 X 1 P M in 7-CD solutions at the following concentrations: (a) 0 M; (b) 0.7 X 10-2 M;(c) 1.5 X M. The inset shows the fitting of X M;(e) 5.0 X experimental points according to eq 5. M; (d) 3.3
i
m
H
0
550
600
Estimated errors are 25%.
650
h,nm Figure 4. Emission spectra of [EB] = 5 X 1 P M in a-CD solutions at the following concentrations: (a) 0 M; (b) 2.5 X 1t2M;(c) 5.0 X M;(d) 7.0X 10-2 M. The inset shows the fitting of experimentalpoints according to eq 5.
of CD the equation simplifies to
Io = ca0 (2) with CD being in large excess with respect to the dye, the molar fraction of complexed and uncomplexed species can be expressed in terms of the equilibriumconstants KAand of the concentration of CD, [CD]
(4)
With division of eq 1 by eq 2 and substitution of eqs 3 and 4, the following expression can be derived
A nonlinear fit to this equation allowsone toderive theequilibrium constant, KA, and the ratios of the quantum yields of complexed and uncomplexed species (@.cD/@o).In the insets of Figures 2 and 3 are reported such plots. Table I1 gives the determined parameters. It must be noticed that given the relatively low association constants of the complexes and the range of concentration which can be explored because of solubility problems,
HzO
4300
110 (95)b
100 (85)b
methanol 4400 460 480 ethanol 4300 610 680 D(+)-glucose 4500 110 (95)b 110 (85)b a-CD 4700 110,280 110 6-CD 4600 120,300 105 7-CD 4700 140,c580 140: 660 0 Determined with time correlated single-photon counting equipment. The value in parentheses is obtained by applying deconvolution to the instrumental response profile. e Average for different concentrationsof y-CD. This lifetime increases with CD concentration. See Discussion section. the plateau region is hardly reached. This introduces large errors in the determined parameters; therefore KAhere reported can be considered in reasonable agreement with those of Table I determined by i d spectra. For a critical examination of these values, see the Discussion section. Fluorescence Lifetimes. The lifetimes of the dyes in water and D(+)-glucose solutions were satisfactorilyfitted to the same single exponential. The luminescence of RB in a- and 8-CDs, and of FL in a-,O-, and y-CDs decays as a single exponential identical to the one measured in neat water. The emissions of EB in a-, P-, and y-CDs and of RB in y-CD could be fitted by a linear combination of two exponentials:
where A1 and A2 stand for the contribution at zero time of the two components with lifetimes 71 and 72. In Table I11 are collected the emission lifetimes of FL, EB, and RB in several neat solvents and saccharide solutions. The values measured here for fluorescence lifetimesin neat solvents compare well with those reported by Rodgerslz but are somehow different from previous determinations.lOJ1 Some of the decays (experimental points and fitted curve) are shown in Figure 5, where a comparison with the laser profile or decay in water is also shown. When the system exhibits a biexponential decay kinetic, the lifetime of the short component ( T ~is, ) in general, close to the one of the compound in neat water and is assigned to the uncomplexed form. The long lifetime (72) is assigned to the complexed form and in y-CD complexes is very similar to the lifetime in ethanol, while in the smaller a- and 8-CDs it is intermediate between thevalue in water and methanol. The ratio A2/A, of the preexponentials is related to the concentration of the two components where C is the concentration, k, stands for the radiative rate constant, and c is the molar absorption coefficient at the excitation wavelength respectively of the complexed (index 2) and aqueous (index 1) form of the dye. Since k, is constant" and €1 = €2, A2/A1= C2/C1. Given the excess of CD with respect to the dye,
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The Journal of Physical Chemistry, Vol. 97, No. 38, 1993
Flamigni
i. 00
Figure 5. Experimental points and computed fits of time profiles of the streak images of the fluorescence decay of the following: (a) EB in water and laser profile at 8.2 ps/channel; (b) EB in [a-CD] = 3.0 X 10-2 M and EB in neat water, 16.3 ps/channel; (c) RB in [y-CD] = 3.3 X 10-2 M RB in neat water, 16.3 ps/channel; (d) RB in [a-CD] = 3.0 X M and RB in neat water, 16.3 ps/channel. A2/A1 can be rewritten: On the basis of the icd spectra and fluorescence data, the Occurrence of inclusion is shown and the presence of at least two different types of complexes is evidenced. "Type 1" complex This equation allows one to estimate KA from time resolved shows a positive sign of the icd band in the visible and has in experiments. In the cases of a- and 8-CD complexes with EB halogenated fluoresceines a lifetime similar to the one of the dye the ratio of the preexponentials allowed calculation of an in ethanol, 580 ps compared to 610 ps for EB and 660 ps compared association constant respectively of 7 and 18 M-I, in reasonable to 680 ps for RB. "Type 2" complex exhibits splitting of the agreement with the association constant determined by the other visible absorption band in icd spectra into a negative and a positive methods. The association constant derived by the preexponential band and in halogenated fluoresceins has emission lifetimes ratio for the systems EB/y-CD and RB/y-CD was concentration intermediate between water and methanol. dependent and by far too low compared to KA calculated by the EB is included by a-,/3-, and y-CDs, the association constant other methods. An interpretation of this inconsistency is given decreases with the dimension of the cavity, and the type of complex in the Discussion section. changes from type 1 in y-to type 2 in a-and &CDs. RB includes Discussion only in the larger y-CD forming the type 1 complex with an association constant similar to that of EB/y-CD systems. From In the present study the interactions of xanthenes with D(+)the similarity of behavior of EB and RB with respect to y-CD, glucose are compared to those with cyclodextrins. The differences the hypothesis is advanced that type 1 complex involves inclusion found are assigned to the interaction of our probes in the ground of the part of the molecule which is common to both dyes, that states with the cavity of the CD to form an inclusion complex. is the xanthenic moiety containing iodine substituents. From a F1, RB, and EB are optically inactive, but their inclusion in the chiral cavity of CD induces optical activity, evidenced by icd comparison of the molecular dimensions to the cavity dimensions, spectra. Moreover, thedifferent signs of the signals give indication it can be deduced that only part of the xanthenic system can be of different orientations of the included molecule with respect to accomodated in the cavity, leaving the rest of the molecule the molecular axis of CD molecule. protruding in water. The hydrogen donating properties of the microenvironment a-CD and 8-CD can associatewith EB and not with RB. Since are reflected by differences in luminescence lifetimes and yields the difference in the two molecules lies in the benzoic acid moiety of RB and EB but not of FL. This is fully consistent with the which in RB is completely substituted with bulky C1, this part solvent dependence of the nonradiative and the solvent invariance of the molecule is believed to enter the cavity in the complexes of the radiative rate constants of the emitting state of these formed by EB with a-and O-CDs, namely, type 2 complexes. In dyes.lO-ll At variance with the halogenated derivatives, F1 RB the substitution by chlorine increases the diameter of the deactivates by radiative processes,'o and it is therefore totally benzoic acid moiety from 5 to 7.5 A and it cannot fit anymore insensitive to the solvent's properties, as shown by the data of Table 111. into the a-and 8-CD cavities. This kind of inclusion leaves the
Inclusion of Fluoresceins in Cyclodextrins
\ w ;
\ii .._
m-cD
w .-,
''type *"complex
"type 1"complex
Figure6. Schematic representation of the two types of complexes for the case of EB.
xanthenicring even moreexposed to water than the type 1complex. A schematicrepresentation of the two complexes for EB is reported in Figure 6. A picture of the two complexes as the one given above also agrees with the different emission lifetimes. The emission parameters of halogenated fluoresceinsare sensitiveto the medium which, affecting the energy splitting between the excited singlet and the triplet, alters the nonradiative rate constant (intersystem crossing) of the emitting state and therefore changes the lifetime.lOJ1 The lifetimes of type 1 complexes are very similar to the emission lifetimes in ethanol, suggesting that the molecule experiences a similar microenvironment. This is in agreement with the data reported by Fleming and co-workers18who found that the basicity of the environment experienced by a CD complexed molecule protruding in water is close to that of ethanol. In other words, the water surrounding the CD rim is less acid than the bulk water. The here reported data, showing a decrease of the hydrogen donating ability of peripheral water with respect to bulk water, are in the same direction. The change in the properties of the water surrounding the dextrins can well be due to the extensive network of OH groups on the edge of the dextrin molecule. The shorter lifetime of the type 2 complex agrees well with a larger protrusion of xanthene ring, on which the excitation is mainly localized, toward a zone where the perturbation effect of the hydroxyl rim tends to decrease and the microenvironment is more similar to bulk water. A perturbation effect limited to a few water solvation shells was also reported in Fleming's data.18 There is practically no effect of solvent on fluorescein luminescenceparameters, as discussed above. The small increase in luminescence yield in concentrated samples is justified by deaggregation of nonluminescentdimers, upon complexationwith CD. Therefore the only informative data for FL/CD systems come from icd spectra. From these it appears that FL, smaller than the halogenated derivatives,tends to associate preferably to &CD, where it can tightly fit, rather than with the larger y-CD, which forms a looser complex. The association constant for FL/ &CD is the largest found for the systemsstudied,and this probably reflects the increased hydrophobicity of the molecule which
The Journal of Physical Chemistry, Vol. 97, No. 38, 1993 9571
possessesless local charge densities than the halogen derivatives. The here found KA (360 M-1) is much larger than the one previously reported7 for the same system (7 M-1) but better compares to thevalues of 2900 M-1 calculated for theassociation constant of the xanthene rhodamine B with b-CD.6 From the signs of icd spectra of FL/CD complexes in thevisible absorption, by analogy to the cases of RB and EB discussed above, the geometry of complexes formed can be deduced. Type 1 complex is probably formed by FL with @- and y-CDs, while FL seems to prefer a type 2 complex with a-CD. In an attempt to rationalize the data, it has been assumed that only one type of complex is formed in each system. This simplification is probably true for a-and &CDs which are too small to accommodate part of the xanthenic ring. In y-CD both types of complexes could in principle be formed. An indication that this is the case is found in the emission lifetime data. In EB/y-CD and RB/yCD systems, the short component, assigned to the uncomplexed dye, is actually longer than the lifetime of the dye in water and tends to increase with the concentration of CD. Furthermore, in the simple biexponential fitting performed here:* the relative weight of the short component (Le. uncomplexed) with respect to the long (i.e. complexed) is higher than expected on the basis of the association constant. All these facts could be explained by the presence of a third component in the decay, which more and more contaminates the short component upon increasing CD concentration. This componentcould be type 2 complex, lessstable than type 1 complex in y-CD systems, but still liable to be formed. The contemporary presence of the two different complexes with different emission yield in y C D systems could also lead to some error in the estimate of KAby the fluorescenceyield method. In particular, since the radiative rate constant of the emitting state is expected to be constant,ll the fluorescence yield of type 1 complex should be about l / 2 that of type 2 complex and this would lead to an underestimate of KA,intended as an overall association constant. On the contrary, the Ac observed in the UV bands of i d spectra for the two complexes does not seems to differ appreciably;23 therefore the KAcalculated by the i d signals treatment could be closer to the overall KA relevant to both equilibria. Conclusions Inclusion of E, RB, and EB with cyclodextrinshas been shown to occur and the association constant has been determined by detecting the effect on ground-state properties (induced circular dichroism spectra) and on thedeactivationof the short lived singlet excited state (fluorescence yield and lifetimes). Two different complexes have been identified on the basis of (i) the signs of their ellipticity in the visible band and (ii) the different emission lifetimes. An hypothesis on the structures of the two complexes has been advanced. In each dye/CD system the formation of only one complex type is generally favored. The Occurrence of the two different complexation types has been detected in y-CD systems which could explain some apparent inconsistency in the results. The use of halogenated fluoresceins as microenvironmental probes has been proven to be effective. The information obtained allows us to assess that the properties of water surrounding the CD rim are modified and resemble those of ethanol. This effect gradually decreases over few solvation shells, in agreement with previous reports.'*
Acknowledgment. The author is indebted to Prof. P. Biscarini of the Department of Inorganic and Physical Chemistry of the University of Bologna for the use of the Jasco 5-500 dichrograph. Stimulating discussions with the colleagues P. Bortolus and S. Monti are acknowledged. The author thanks L. Ventura, M. Minghetti, and R. Chiodini for technical assistance and G. Gubellini for the drawings.
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References and Notes (1) Bender, M. L.; Komiyama, M. Cyclodexfrin Chemistry; Springer Verlag: Berlin, 1978. (2) Szejtli, J. Cyclodexrrinr and their inclusion complexes; Akademiai Kiado: Budapest, 1982. (3) Saenger, W. Angew. Chem., Inr. Ed. Engl. 1980, 19, 344. (4) Flamigni, L. J. Phys. Chem. 1992, 96,3331. (5) Politzer, I. R.; Crago, K. T.; Hampton, T.; Joseph, J.; Boyer, J. H.; Shah, M. Chem. Phys. Lerr. 1989, 159, 258. (6) Degani, J.; Willner, I.; Haas, Y . Chem. Phys. Leu. 1984, 104, 496. (7) Brittain, H. G. Chem. Phys. Lert. 1981, 83, 161. (8) (a) References quoted in ref 4. (b) Neckers, D. C. J. Chem. Educ. 1987,64,649. (c)Paczkowski,J.;Lamberts,T. J. M.;Pacszowska, B.;Neckers, D. C. J. Free Radicals Biol. Med. 1985, I , 341. (d) Sarna, T.; Zajac, J.; Bowman, M. K.; Truscott, T. G. J. Photochem. Phorobiol. A : Chem. 1991, 60, 295. (9) Neckers, D. C.; Paczkowslci, J. J. Am. Chem. Soc. 1986,108,291. (10) Fleming, G. R.; Knight, A. W. E.; Moms,J. M.; Morrison, R. J. S.; Robinson, G. W. J. Am. Chem. SOC.1977, 99,4306. (11) Cramer, L. E.; Spears, K.G. J . Am. Chem. SOC.1978, 100, 221. (12) Rodgers, M. A. J. Chem. Phys. Lerr. 1981, 78, 509. (13) Reed, W.; Politi, M. J.; Fendler, J. H.J.Am. Chem. Soc. 1981,103, 4591.
Flamigni (14) Rodgers, M. A. J. J. Phys. Chem. 1981.85, 3372. (15) Cox, G.S.;Hauptmann, P. J.; Turro, N. J. J . Phorochem. Phorobiol. 1984, 39, 591. (16) Heredia, A.; Requena, 0.;Sanchez, F. G. J. Chem. Soc., Chem. Commun. 1985, 24, 1814. (17) Eaton, D. Tetrahedron 1987,43, 1551. (18) Hansen, J. E.; Pines, E.; Fleming, G.R. J. Phys. Chem. 1992, 96, 6904. (19) Alchemy I& T r i p Associates Inc.; St. Louis, MO, 1988. (20) Benesi, H. A.; Hildebrand, J. H. J. Am. Chem. Soc. 1949,71,2703. (21) Monti, S.Private communication. (22) The attempt to fit the fluorescencedecays with more complex models was unsuccessful mainly because of the noise level and distribution in a (basically) single shot experiment as those which can be produced by our instrumentation. (23) Thiscan bederivedfrom FigureZa4,performedat theconcentration of EB = 9.5 X l e 5 M and respectively a-CD = 3 X 10-2 M, ,%CD = 1.4 X 10-* M, y C D = 2.1 X l e 2 M. By using KA of Table I and the optical path used in the experiment, a Ac of ca. 20 OOO mdeg mol-’ cm-I is derived for the cases of a-and &CD complexes with EB and a Ac of ca. 26 OOO mdeg mol-l cm-I for the y-CD complex with EB.
