Influence of double confinement on photophysics of 7-(diethylamino)coumarin-3-carboxylic acid in water/AOT/isooctane reverse micelles†

The effect of double confinement on the photophysics of 7-(diethylamino)coumarin-3-carboxylic acid (7DCCA) inside the water pool of water/AOT/isooctane reverse micelles has been reported in this study. At first a supramolecular host–guest complex was formed in water between 2-hydroxypropyl-gcyclodextrin (HP-g-CD) and 7-DCCA. Then the aqueous solution of this complex was used to form reverse micelles at any particular w0 value (w0 1⁄4 [water]/[surfactant]). We have used sodium dioctyl sulfosuccinate (AOT) as surfactant and isooctane as non-polar solvent to prepare reverse micelles. A comparative study between double confinement system and the single confinement system, where the 7-DCCA molecule was incorporated inside the core of the water/AOT/isooctane reverse micelles, was done. We have used the steady state absorption and fluorescence emission techniques to highlight the significant shift of the spectral behaviour of the 7-DCCA due to the double encapsulation of the dye in the nanopool of the reverse micelles. More affirmation has been achieved by the use of time resolved fluorescence emission spectroscopy. The study of solvation dynamics and rotational relaxation dynamics was used as tools to investigate the effect of double encapsulation on the excited state dynamics of the probe molecule. These excited state dynamics clearly show that even at the highest w0 value studied here, the excited state dynamics of the doubly confined dye are significantly different from those of the single confined dye in the reverse micelle. The higher values of fluorescence emission decay time, rotational relaxation and solvent relaxation times in the doubly confined system compared to those of the single confined system at different w0 values proved the existence of the supramolecular host–guest complex inside the core of the water/AOT/isooctane reverse micelle.


Introduction
5][6][7] The photophysical properties of 7-aminocoumarins are dependent on the polarity and viscosity of the media and on the specic solute solvent interaction with the media.These substituted amino coumarin dyes form an intramolecular charge transfer (ICT) state when excited in a polar medium.][10][11] This TICT state formation is essentially a nonradiative process.These TICT states may be emissive or non emissive in nature.3][14] The solvation dynamics in different conned medium like micelles, reverse micelles etc. are much slower than that in neat solvent, due to the restriction imposed on the solvent molecules in these medium.Here in this study we have used reverse micelle as the conned medium.Reverse micelles are the aggregates of surfactant molecules in nonpolar solvents, where the polar or ionic head groups of the surfactants point towards the polar solvent pool and the long nonpolar hydrocarbon chain pointing towards the nonpolar solvents. 15,16Aqueous reverse micelles facilitate the solubilisation of sufficient amount of water in the core. 17,18ater/AOT/isooctane reverse micelle is a ternary system composed of water, Aerosol OT (AOT) surfactant and isooctane (oil).0][21][22][23] The water molecules present in the water pool of the reverse micelles are different in behaviour compared to bulk water.5][26][27][28][29][30][31] Gorski and Ostanevich using the small-angle neutron scattering reported several important features of AOT reverse micelles. 32,33All the water molecules in the core, except six water molecules per AOT molecule freeze at understand the modication and improvement of photophysical properties of a hydrophilic and TICT forming dye (like 7-DCCA) upon a double connement in the reverse micelles.For this purpose we have used the uorescence emission properties of 7-DCCA as tool.The change in emission quantum yields, solvent relaxation, rotational relaxation of 7-DCCA upon double connement was used as experimental evidences.4][45][46] We have studied the effect of single connement on the photophysical properties of 7-DCCA in the nano-pool of water/AOT/isooctane reverse micelles of different sizes and reported the excitation wavelength dependent photophysics.We had also demonstrated the red edge excitation shi in the core of reverse micelle. 47In this study we have shown the double connement of a hydrophilic molecule 7-DCCA in the core of the reverse micelles and its effect on the uorescence emission properties.We have compared our result with our previous study, 47 where we had demonstrated the effect of single connement on the photophysics of 7-DCCA in the reverse micelles.

Materials and methods
7-DCCA was purchased from Sigma-Aldrich and used as received.Sodium dioctylsulfosuccinate (AOT) was purchased from Sigma-Aldrich.AOT was dried under vacuum and used as previously described in literature.For all the experiments the concentration of AOT has been maintained at 0.1 (M).Isooctane was purchased from Spectrochem, India.The required amount of water has been added to the system to form the reverse micelles at different w 0 values, where w 0 is represented as [water]/[AOT].2-Hydroxypropyl-g-cyclodextrin (HP-g-CD) was purchased from Sigma-Aldrich and used as received.For the preparation of aqueous solution of 7-DCCA and reverse micelles Millipore water was used.For the double connement of the molecule, we have used the procedure described by Jane et al. 41 For the host guest complexation in neat water, the concentration of 7-DCCA and HP-g-CD was maintained at 5.5 Â 10 À4 (M) and 6 Â 10 À2 (M).During the double connement studies the dye concentration is maintained at 3 Â 10 À6 (M).The nal concentration of HP-g-CD during this double connement studies is maintained at 3.2 Â 10 À4 (M).UV-Vis absorption studies were carried out using UV-Vis spectrophotometer (model: UV-2550, Shimadzu).The steady state uorescence emission measurements were executed using a Fluoromax-4P spectrouorometer (Horiba Jobin Yvon).In the case of steady state absorption and uorescence measurement the temperature was kept constant at 298 K by using a Jeiotech refrigerated bath circulator (model: RW0525G).The uorescence quantum yields of 7-DCCA + HP-g-CD complex in different reverse micellar media were measured using the uorescence quantum yield of Coumarin 480 in water solution (f f ¼ 0.66) as reference, 48 by using the following equation: where s and r stand for the sample and reference respectively.
Here I stands for the integrated area under the uorescence curve, A stands for the absorbance of the sample at excitation wavelength and n stands for the refractive index of the medium.
The uorescence time resolved decays were measured by using a picosecond time-correlated single-photon counting (TCSPC) technique.We have used a time-resolved uorescence spectrophotometer from Edinburgh Instruments (model: Life-Spec-II, U.K.) for lifetime measurement.We have used picosecond diode lasers with excitation wavelength at 405 nm.The full width at half maximum (FWHM) of our system is $90 ps.The uorescence transients were detected at magic angle (54.7 ) polarization using Hamamatsu MCP PMT (3809U) as a detector.The decays were analysed by using F-900 decay analysis soware.The uorescence anisotropy decay (r(t)) was measured by using the same instrument.The following equation was used to obtain r(t).
where the emission intensities at parallel (I k ) and perpendicular (I t ) polarizations were collected alternatively by xing the time for both the decays.We had used motorised polarizers to collect the parallel and perpendicular decays.G is the correction factor for the detector sensitivity to the polarization direction of the emission.Similar method was used to measure the G factor.F-900 soware was used to analyze the anisotropy decay.
The overall anisotropy decay is represented by the following equation which is the sum of two exponential equations: 49 For the time resolved measurements the temperature was kept constant at 298 K by using Peltier-controlled cuvette holders from Quantum Northwest (model: TLC-50).
In order to study the solvation dynamics we have constructed time resolved emission spectra (TRES) according to the procedure described in the literature. 50,51The spectrum at any time (t) is represented as S(l; t) and is describe as follows: Sðl; tÞ ¼ Dðt; lÞ s 0 ðlÞ ð N 0 Dðt; lÞdt (5)   where D(t; l) is representative of tted decays.Thus S(l; t) can be easily obtained from the tted decays D(t; l) by the relative normalisation of the steady state emission spectrum.All TRES are tted by using the log-normal function: where n p , I 0 , D, and b stand for peak frequency, peak height, width parameter and asymmetric parameter, respectively.To get the solvent relaxation time we have deduced the solvent response function [C(t)]: n(t), n(0), n(N) are the peak frequencies at the time t, at the time t ¼ 0 and at t ¼ N. Now, the decay of C(t) with time can be tted by the exponential function: where a i stands for the amplitude and s i stands for the solvent relaxation time constant.The average solvation time can be expressed using the following equation:

Result and discussion
3.1 Steady state absorption and emission spectral studies 7-DCCA is an acidic dye remaining in the water solution as a deprotonated species. 45It shows a prominent and single absorption peak at 409 nm in water.We have studied the steady state absorption spectral feature of double conned 7-DCCA in the CD/water/AOT/isooctane reverse micelles and compared this result with the single conned system.We had reported earlier the spectral properties of 7-DCCA dye in water/ AOT/isooctane reverse micelles. 47We have seen that the absorption peak of 7-DCCA in CD/reverse micelle at w 0 ¼ 3 appears at 391 nm and with gradual swelling of the reverse micelle core, the absorption peak undergoes red shi, thereby reaching 399 nm at w 0 ¼ 30 as shown in Fig. 1 and Table 1.
This absorption peak at w 0 ¼ 30 is blue shied by 7 nm compared to 7-DCCA dye in water/AOT/isooctane reverse micelles.This shows the effect of double connement of 7-DCCA on the ground state spectral property inside the reverse micelle.For every w 0 value the absorption peak of doubly conned dye is blue shied compared to the singly conned dye in the same reverse micelle.In our present system the absorption peak is red shied from 391 nm at w 0 ¼ 3 to 399 nm at w 0 ¼ 30 whereas, the absorption peak underwent red shi from 394 nm at w 0 ¼ 3 to 406 nm at w 0 ¼ 30 for singly conned dye in the water/AOT/isooctane reverse micelles. 47This clearly indicates that due to double connement 7-DCCA experiences quite different environment than singly conned 7-DCCA in the same reverse micelles.These absorption peaks however, are blue shied compared to the absorption peak of 7-DCCA in neat water (Fig. 1).This clearly indicates that 7-DCCA experiences certainly a different atmosphere inside the CD/reverse micelle due to double connement.We can affirmatively say that the 7-DCCA resides completely inside the water pool of the reverse micelles because of two reasons (a) 7-DCCA is highly soluble in water and (b) being complexed with HP-g-CD it becomes more soluble in water.So it is expected that 7-DCCA + HP-g-CD complex will reside completely inside the water pool of the reverse micelle.When we have studied the host guest complexation of 7-DCCA ([7-DCCA] ¼ 3 Â 10 À6 (M)) with HP-g-CD we had found that the absorption peak of 7-DCCA underwent blue shi from 409 nm in water to 403 nm in the presence of 7.30 mM HP-g-CD.So, further blue shi of the absorption peak in case of doubly conned dye clearly demonstrates the changing environment inside the reverse micelle due to greater connement.7-DCCA is poorly soluble in isooctane and shows three absorption bands located at 413 nm, 402 nm and 390 nm.Coumarin 343 shows mainly two absorption bands in n-heptane at $402 nm due to monomer and $428 nm due to aggregated form. 527-DCCA also shows similar feature corresponding to monomer absorption at 390 nm and aggregated absorption at 413 nm in isooctane.In our case, the absorption band at w 0 ¼ 3 appears at 391 nm, so we are expecting the emission from the monomer form of the dye, which is highly soluble in water (Fig. S1 †).
We have compared the uorescence emission properties of 7-DCCA in CD/reverse micelles with the emission spectra of 7-DCCA in the reverse micelles.We have found that for all the w 0 values the uorescence emission peak of 7-DCCA in doubly conned system (l exi ¼ 405 nm) is red shied compared to that of 7-DCCA in singly conned system.The uorescence emission peak of 7-DCCA in doubly conned system appears at 453 nm at w 0 ¼ 3 and with gradual swelling of the reverse micelle core the uorescence emission peak gets red shied to 462 nm at w 0 ¼ 30 (Fig. 2 and Table 1).In our previous study we have observed that 7-DCCA inside the reverse micellar core shows its emission peak at 450 nm at w 0 ¼ 3 and 454 nm when w 0 ¼ 30. 47This clearly indicates that for all the w 0 values the emission peak of 7-DCCA in doubly conned system inside the core of the reverse micelle is red shied compared to the singly conned dye inside the reverse micelle.Moreover, 7-DCCA in water solution shows a prominent emission peak at 470 nm.When encapsulated by HP-g-CD it shows emission band at 461 nm.When this complex is trapped further inside the core of water/AOT/isooctane reverse micelles, we have found that the emission peak undergoes further blue shi to 453 nm at w 0 ¼ 3. Probably, this is due to the less polar environment faced by the doubly conned dye inside the core of reverse micelle.We know that the water molecules present inside the core of reverse micelle are less polar compared to the bulk water molecules. 53,54With the gradual addition of water in the reverse micelles the uorescence emission peak gradually undergoes red shiing as the polarity of the water pool increases.Moreover, when the dyemacrocycle complex is entrapped inside the core of reverse micelle the dye faces more restricted environment inside the core of reverse micelle.This is due to the fact that the viscosity of water inside the core of reverse micelle is higher than the bulk water and this viscosity gradually decreases with the gradual increase of water content. 55So the high viscosity of water inside the reverse micelle core at lower w 0 value imposes greater restriction to the doubly conned dye to form TICT state, thereby causing the blue shied emission peak in the reverse micelle.This high restriction imposed on the 7-DCCA due to double connement in the core of reverse micelle causes a high quantum yield of the dye (Table 1), compared to single connement of dye in the same reverse micelle. 47The uorescence quantum yield of the doubly conned dye gradually decreases from 0.59 at w 0 ¼ 3 to 0.15 at w 0 ¼ 30, demonstrating that with gradual swelling of the water pool, the dye-macrocycle complex experiences gradually decreasing restriction, thereby increasing the tendency to form TICT state by the rotation of diethylamino group in the excited state (l exi ¼ 405 nm).This shows that the dye experiences greater restriction imposed on it due to double connement than the single connement inside the reverse micelle.This difference in uorescence quantum yield value is prominent upto w 0 ¼ 10, between doubly conned Fig. 1 The change of the absorption spectral peak position of 7-DCCA in the doubly confined system in the reverse micelles with change in w 0 from 0 to 30 and in neat water.
Table 1 The photophysical parameters of 7-DCCA in the doubly confined system in the water/AOT/isooctane reverse micelles at different w 0 values dye and the singly conned dye.Beyond w 0 ¼ 10 the difference gradually starts to decrease.At w 0 ¼ 20, the uorescence quantum yield is 0.17 for the doubly conned dye, the same is 0.15 for singly conned dye inside the reverse micelle.At w 0 ¼ 30 the quantum yield values are almost same for both the cases.7-DCCA exhibited very low solubility in nonpolar solvent.In isooctane it shows the emission spectrum which is quite similar in shape as that of Coumarin 343 in cyclohexane.Correa and Levinger reported the emission spectra of Coumarin 343 in cyclohexane and reported the emission from both the monomer and aggregated form. 56A similar type of emission feature is observed in the case of 7-DCCA in isooctane.As in the present experimental condition, the dye is complexed with HP-g-CD and the dye concentration in the reverse micelle is sufficiently low so the aggregate formation is inhibited.Moreover, the emission spectra of the dye-macrocycle complex inside the reverse micelle at w 0 ¼ 3 is sufficiently red shied compared to the emission bands of 7-DCCA in isooctane indicating that the dyemacrocyclic complex residing in the water pool and not in the nonpolar solvent (Fig. S2 †).At w 0 ¼ 3, the dye-macrocycle complex is expected to reside near the interface and experiences very high restriction, making its uorescence quantum yield value very high compared to that of singly conned dye in the reverse micelle at same w 0 .
It is known that the addition of cyclodextrin (HP-g-CD) and 7-DCCA can cause the perturbation of the reverse micelle and the properties of reverse micelles are not like before. 41We have previously seen that 7-DCCA shows emission spectra at 470 nm in bulk water and when complexed with HP-g-CD shows a blue shied emission at 461 nm.Similarly, at w 0 ¼ 3 it is observed that doubly conned dye shows a red shied emission compared to the singly conned dye in the same reverse micelle at w 0 ¼ 3. Again this red shi increases with the increase of w 0 value and this red shied emission for the dye-macrocyclic complex is highest at w 0 ¼ 30.This red shied uorescence emission peak of 7-DCCA in doubly conned system compared to singly conned system in the same reverse micelle at different w 0 values are clear evidence of interaction of dye with macrocycle inside the core of reverse micelles.Similar kind of observation was observed by Jane et al. who showed the interaction between a hydrophobic dye Coumarin 522 with comparatively less water soluble b-cyclodextrin. 41They reported three probable types of formation of supramolecular host-guest complex inside the core of the reverse micelle.The rst assumption dealt with the formation of unperturbed complex between the dye and macrocycle.Since the water pool at the core of reverse micelle is perturbed aer the addition of solutes, this kind of unperturbed host guest complexation in the water pool of reverse micelle is unlikely to take place.A second assumption, which says minimal interaction between the dye and the macrocycle is ruled out, as here both the dye and the macrocycle HP-g-CD are sufficiently hydrophilic in nature.In our system, the red shiing of emission spectra of the doubly conned dye compared to singly conned dye inside the core of reverse micelles ranges from 3 nm at w 0 ¼ 3 to 8 nm at w 0 ¼ 30.These further rules out the second assumption of minimal interaction between the dye and the macrocycle inside the water pool of the reverse micelles.The third assumption deals with the formation of the dye-supramolecular complex inside the core of reverse micelle in the perturbed water pool.Here the dye-HP-g-CD complexes exist in the water pool inside the reverse micelle.In our case, the red shied emission spectra compared to the emission spectra of singly conned dye inside the reverse micelle at every w 0 value provides denite proof of the formation of a supramolecular host-guest complex inside the core of reverse micelle.Now we need the explanation of red shied emission spectra of dye in the doubly conned complex inside the core of reverse micelle compared to that of single connement dye inside the same medium.The two simultaneous and complementary processes inside the cavity of reverse micelles can also account for the observed red shi.Jane et al. provided the idea of these two processes; one of which is the trapping of both dye and macrocycle inside the cavity of reverse micelle and second being the perturbation of the reverse micelle by both the dye and macrocycle, thereby increasing the polarity of water trapped in the core of reverse micelles. 41The water molecules trapped inside the cavity of HP-g-CD may also contribute to the observed red shi.Now perturbation of the reverse micelle by trapping both the dye and the HP-g-CD causes the greater mobilisation of the water molecules inside the cavity of the cyclodextrin, thereby causing the increase of polarity inside the cavity of HP-g-CD and red shiing the emission peak compared to that of pure dye inside the water reverse micelle.With the gradual swelling of the reverse micelle the number of free water molecules increases.These are more polar than the bound water molecules and mobilisation of these free water molecules inside the cavity of HP-g-CD causes the greater red shiing of the emission spectra compared to that of singly conned dye in the same medium.Again, the existence of a supramolecular host-guest complex in the water pool of the reverse micelle is evident from the clear difference of full width at half maximum (FWHM) of dye in doubly and singly conned systems.In our present system the FWHM values decreases in a regular pattern whereas, in case of singly conned dye in the core of reverse micelle FWHM increased in a continuous way.The difference of FWHM values for doubly and the singly conned dye inside the reverse micelle at every w 0 value indicates the existence of a dye macrocycle complex in the core of reverse micelle.The lower FWHM for our present system compared to that of single connement dye in water/AOT/ isooctane reverse micelles 47 depicts greater connement of the dye in our present system.

Time resolved emission spectral studies
We studied the time resolved emission spectra (l exi ¼ 405 nm) in order to have a better understanding about the double connement of dye inside the reverse micelle.We tted all the decay proles by biexponential function.We found that at w 0 ¼ 3 the uorescence emission decay consists of two components, one of which is of 1.313 ns (43%, fast component) and another one being 2.553 ns (57%, slow component).With the gradual increase of water pool size inside the core of reverse micelle we have found that the fast component decreases in timescale but at the same time its weight percentage is gradually increasing.It is found that the timescale value for the fast component gradually decreases from 1.313 ns at w 0 ¼ 3 to 0.552 ns at w 0 ¼ 30.The weight percentage of this component gradually increases from 43% to 94%.On the other hand, the slow component also gradually decreases in its timescale from 2.553 ns to 1.893 ns.The weight percentage of this component also gradually decreases from 57% at w 0 ¼ 3 to 6% at w 0 ¼ 30 (Fig. S3 † and Table 2).This indicates the gradual relaxing environment around the dye-macrocycle complex with gradual swelling of the water pool.This decrease in restriction around the dye-macrocyclic complex is also reected from the decrease of average decay time with the gradual increase of w 0 value (Fig. 3(a) and Table 1).This gradual decrease in average uorescence decay time is mainly due to the gradual decrease of restrictedness of the reverse micellar core, causing the increase of the non radiative decay pathway.Now we have found in our system that for w 0 ¼ 3 the uorescence decay time of 7-DCCA is greater for doubly conned complex than the singly conned dye inside the water/AOT/isooctane reverse micelle at the same w 0 value.When the water pool is small enough at w 0 ¼ 3, the semi frozen nature of the water molecules due to the presence of a predominantly large number of bound water molecules causes the greater restriction and greater connement of 7-DCCA inside the pool.This causes the greater uorescence decay time of dye in doubly conned system compared to that in singly conned system in the same medium.With the gradual increase of size of the water pool inside the core of reverse micelle the number of free water molecules increases causing the greater mobilisation and greater polarity of water.This gradually releases the restriction and the dye-macrocycle complex experiences comparatively less connement.Even at w 0 ¼ 30 the uorescence decay time of 7-DCCA in doubly conned system is higher than that single conned system in water/AOT/isooctane reverse micelle at w 0 ¼ 30.This clearly showed that even at very high w 0 value, the dye experiences greater restricted environment in doubly conned system compared to single conned system at the same w 0 ¼ 30 value.This clearly demonstrates the probability of interaction between the dye 7-DCCA and macrocycle HP-g-CD inside the core of reverse micelle in the conned water pool.We have found out the partition coefficient of 7-DCCA when complexed with HP-g-CD in water to know the percentage of complex or free dye present in the solution, when adding water solution of dye-macrocyclic complex in the isooctane-AOT mixture.This will also help us to know the amount of dye formed complex with HP-g-CD in the water solution used to form the reverse micelles.We have used the following equation for nding out the partition coefficient of dye in between water and the macrocycle HP-g-CD: 57,58 where, s, s W , s CD are the uorescence average decay time, uorescence decay time in water, uorescence decay time of 7-DCCA bound to HP-g-CD.K p and g CD stand for partition coef-cient and the molar volume of HP-g-CD.From the plot of s against [CD] we have found that the partition coefficient is 1.36 Â 10 2 together with g CD value 1.36 dm 3 mol À1 .The plot gives a very good tting with the tting parameter R 2 ¼ 0.997 (Fig. S4 †).The large partition coefficient value indicates a very strong binding of the dye with the macrocycle HP-g-CD in water and it is observed that about 99.3% dye is complexed with the macrocycle in water, leaving very small amount of un-complexed dye in water.So most of the dye encapsulated inside the core of reverse micelle as complex with HP-g-CD.We have previously mentioned that this dye-macrocycle complex is quite different in nature inside the water pool of the reverse micelle than in the bulk water as evident from the uorescence decay time.Moreover, in bulk water the decay of 7-DCCA complexed with HP-g-CD is tri-exponential in nature, whereas that inside the core of reverse micelle is completely biexponential in nature.This also clearly demonstrates the difference of nature of complex of 7-DCCA with HP-g-CD in the water pool of reverse micelle and bulk water.Initially, when the pool is small in size the dye-macrocycle complex mainly resides near the interface.With the gradual increase in size of the water pool it shows tendency to move towards the centre of the pool, because the dye 7-DCCA has been made more hydrophilic by encapsulating it inside the cavity of highly water soluble macrocycle HP-g-CD, so that no portion of dye resides in the nonpolar phase.This ensures that the dye-macrocycle complex completely reside inside the core of reverse micelle in water pool.With the gradual increase of the pool size (i.e. with increasing w 0 value) the dye-macrocycle complex shows greater propensity to move towards the core of the pool from the interfacial region.Since, the complex is more hydrophilic than the uncapsulated dye and in the large reverse micelles the number of free water molecules increases, thereby increasing the polarity of the core of the water pool.For the dye 7-DCCA, TICT formation is the main nonradiative deactivation pathway and this deactivation goes through the formation of ICT state, and it further undergoes ultrafast twisting motion to produce TICT state.The rate constant of radiative and non radiative deactivation pathway is provided by the following equations: Comparison of the values of radiative and non radiative decay rate constant, between the doubly and singly conned molecules, helps us to understand the nature of greater connement of the dye in our present system.The non radiative decay rate constant (k nr ) in case of singly conned dye is always higher than doubly conned dye.It is observed that in water aer addition of 3.41 Â 10 À4 (M) HP-g-CD to the aqueous solution of 7-DCCA, the non radiative decay rate constant become 5.68 Â 10 9 s À1 .This shows that in double connement system the restrictedness around the dye is higher compared to both single connement system and in supramolecular host guest complex in water.This shows that in doubly conned system the greater restrictedness prevents the formation of TICT state so that the nonradiative decay constant becomes signicantly lower.We have found that at w 0 ¼ 3, the value of k nr is 0.203 Â 10 9 s À1 and this constant increases to 1.345 Â 10 9 s À1 at w 0 ¼ 30 (Fig. 3(b) and Table 1).This clearly depicts that with the gradual increase of the water pool, the restriction on the dye-macrocycle complex inside the core of the reverse micelle decreases.With the gradual increase of the water pool size the polarity of the water pool increases as polarity of the water pool is directly related to the w 0 value. 59,60oreover, with the gradual swelling of the water pool the viscosity of the pool decreases.So with the gradual rise of size of the water pool the more polar water molecules enter inside the cavity of the cyclodextrin HP-g-CD causing nonradiative deactivation of the excited state.Moreover, decreasing viscosity of water with the gradual increase of the w 0 value increases the nonradiative decay rate of the dye entrapped inside the cavity of the HP-g-CD.The formation of the dye-macrocycle complex inside the core of reverse micelle is also evident from the value of k nr , it is lower for our present system than that of singly conned dye in the same system at all the w 0 values. 47This clearly indicates that the dye is undergoing interaction with the macrocycle inside the core water/AOT/isooctane reverse micelles.It is found that for doubly conned system at w 0 ¼ 3 the k nr value is 0.203 Â 10 9 s À1 whereas, at w 0 ¼ 3 for singly conned dye inside the reverse micelle core 47 k nr value is 0.335 Â 10 9 s À1 .This shows greater connement of the dye in the doubly conned system.Even at w 0 ¼ 30 the k nr value of doubly conned dye is lower than the single conned dye inside the same system.This clearly demonstrates that even at w 0 ¼ 30 the interaction between the dye and the macrocycle persists in the water pool.

Solvation dynamics
We have studied the solvation dynamics in our present system.We found that for our present system, the decay of C(t) was tted biexponentially upto w 0 ¼ 10 and single exponentially beyond w 0 ¼ 10 (Fig. 4 and Table 3).The time resolved emission spectra (TRES) was constructed for obtaining an idea about the dynamic Stokes shi or time dependent Stokes shi (TDSS).The time resolved emission spectra (TRES) demonstrating the time dependent Stokes shi (TDSS) at two different w 0 values are presented in Fig. S5.† The emission wavelength dependent emission decays ranging from red end to the blue end of the emission peak at two different w 0 values are shown in Fig. 5.We have found that the average solvent relaxation time gradually decreases with the gradual increase of size of the water pool (Table 3).This is due to the increase of free water molecules with the gradual increase of water pool size.We have found that at w 0 ¼ 3 the solvent relaxation time consists of two components.One of those components is slow and the other one is fast.The time scale of the fast component is 0.190 ns, this contributes 34% to the total solvent relaxation, and the slow component of 1.570 ns contributes 66% of the total solvent relaxation.The most important aspect of this solvent relaxation is that the timescale of the fast component is higher than that in the case of singly conned dye in reverse micelle at w 0 ¼ 3, although the weight percentage of the fast component is less than that our previous experiment at same w 0 value. 47The timescale of the slow component and the corresponding weight percentage in our present system is higher than the singly conned dye in the reverse micelles.This result is quite obvious from the point of view that the water molecules inside the pool of reverse micelle enter inside the cavity of macrocycle HP-g-CD.
As a result the restricted and immobilised nature of water molecule increases.This causes the increase of the timescale of both the fast and slow components.The weight percentage of the slow component is higher in the present system compared to singly conned system at w 0 ¼ 3.This conrm our previous assumption that initially at w 0 ¼ 3 (where, the pool size is sufficiently small) the dye macrocycle complex use to reside near the interfacial region.The solvent relaxation time consists of two components upto w 0 ¼ 10, aer that the solvent relaxation time consists of only one component as like singly conned system.Upto w 0 ¼ 5 the average solvent relaxation time is signicantly high for our present system compared to that in singly conned system.Here, the slow component is higher in time scale than that in singly conned dye in the same revere micelle, although the weight percentage is slightly lower than that in singly conned dye.The slow component is due to the   relaxation of the bound water.This demonstrated that the dyemacrocycle complex is moving towards the core of the reverse micelles with the increase of size of the water pool.This is also supported by the fact that both the bound and free water molecules are in dynamic equilibrium and undergoes continuous exchange with each other.So the dye-macrocycle complexes staying near the interfacial region move towards the core of the pool with the gradual swelling of the reverse micelles.Aer w 0 ¼ 5, the average rotational relaxation time gradually starts to become comparable with singly conned medium.We have previously said that the addition of both the HP-g-CD and dye causes the perturbation of the water pool.So this perturbation causes the greater mobilisation of the water molecules inside the cavity of HP-g-CD thereby making the solvent relaxation fast.At w 0 ¼ 10 the fast component is higher in timescale than that in singly conned dye in reverse micelle and also the weight percentage is higher.This shows that free water molecules are moving inside the cavity of the cyclodextrin thereby increasing the timescale.Beyond the w 0 ¼ 10, the solvent relaxation time consists of only one component and this component is almost same as that in single conned system.This indicates that the supramolecular dye-macrocyclic complex has moved towards the centre of the pool, where only one kind of water molecule undergoes relaxation.However, in our case we have found that the dynamic Stokes shi or TDSS value is lower for every w 0 than that in 7-DCCA/water/AOT/isooctane.This also demonstrates the presence of 7-DCCA-HP-g-CD complex inside the core of reverse micelle and also indicates that even at w 0 ¼ 30 this complex persists in the water pool.
Here in order to quantify the energetic associated with the solvation process we have found out the values of DG s using the Eyring equation: 61,62 where DG s is the free energy change of the solvation process, k B and h are the Boltzmann constant and Plank's constant respectively.Here k stand for rate constant for solvent relaxation process.Using this equation we have found that the free energy change of the solvation process gradually changes from 5.21 kcal mol À1 at w 0 ¼ 3 to 4.07 kcal mol À1 at w 0 ¼ 30 (Table 3).Here DG s ¼ (DG* + DG 0 ), where DG* and DG 0 stand for the activation energy of desorption from the surface of the biomolecule and the excess hydrogen bond energy of water with the biomolecule (over that in the bulk).According to the model proposed by Nandi and Bagchi we have taken the value of DG* to be constant (1.5 k B T).As the DG* value is very small, the contribution of DG* in DG s can be neglected.Then the value of DG s is almost equal to DG 0 .Pal et al. had studied the femtosecond dynamics of bulk water and calculated the Gibbs free energy of solvation.They found the free energy to be $750 cal mol À1 . 63However, in our present system we have found that the DG s value ranges from 5.21 kcal mol À1 at w 0 ¼ 3 and 4.07 kcal mol À1 at w 0 ¼ 30.These are much larger than that in water clearly depicting the rupture of hydrogen bonded network of water inside the water pool of reverse micelle.We had found that for all the w 0 values especially for 3 and 5 there are clear differences of DG s values of our present system with single conned system.Aer w 0 ¼ 5 for both the systems DG s values become almost same.This further conrms our previous assumption that the addition of both HP-g-CD and 7-DCCA perturbs the water pool of the reverse micelle and this perturbation causes the difference in average solvent relaxation times in our present system and with that of single conned system.

Red edge excitation shi
We reported the red edge excitation shi (REES) of 7-DCCA inside the core of water/AOT/isooctane reverse micelles at different w 0 values. 47In our present system, we have also found REES.We have found that at w 0 ¼ 3, with changing the excitation wavelength from 405 nm to 435 nm, the emission peak shis by 6 nm.With the gradual increase of the size of the water pool of reverse micelle the REES value gradually decreases as shown in Table 1 and Fig. 2(b).At w 0 ¼ 20 with changing l exi from 405 nm to 435 nm the REES value is only 1 nm, whereas for singly conned system the observed REES is 5 nm for changing the l exi from 405 nm to 425 nm.This indicates a different type of environment faced by the dye inside the core of reverse micelle at even w 0 ¼ 20 in the doubly conned system.This also shows that the dye is buried inside the cavity of HP-g-CD in the core of reverse micelle.This causes the signicantly less REES in our present system compared to singly conned system.In singly conned system, we have found that the extent of REES gradually decreases with gradual swelling of the water pool inside the core of reverse micelle.REES is a technique; it provides valuable information about the relative rate of solvent relaxation.Moreover, this technique helps us to directly monitor the microenvironment and dynamics around the uorophore in different restricted and microheterogeneous medium.There have been reports of observing REES in water reverse micelle by different groups. 56,64-67REES is prominently observed for a number of uorophores in different media like frozen media, in different vitried solution, highly viscous solution and polymer matrices.In our present study, we have seen that, the solvent relaxation time is smaller than the uorescence decay time (s f ) of the uorophore at every w 0 value.This may be due to the specic solute-solvent interaction between the doubly conned Fig. 6 The overlay of time resolved anisotropy decays of 7-DCCA in the doubly confined system at different w 0 values.
dye and water molecule inside the core of reverse micelle.This type of hydrogen bonding interaction is quite common phenomenon for 7-DCCA with polar protic solvent molecules.This substantially modulates the photophysical behaviour of 7-DCCA. 48So this specic solute solvent interaction is mainly responsible for the observed REES in our present system.The water molecules trapped inside the core of reverse micelle act as hydrogen bond donor, whereas 7-DCCA acts as hydrogen bond acceptor.Samanta and co-worker reported similar kind of phenomenon of specic solute solvent interaction between the dye HNBD and RTIL, where the RTIL acts as hydrogen bond acceptor facilitating the photoselection. 68This causes the REES of HNBD in RTIL.With the gradual increase of size of the water pool the extent of red edge excitation shi gradually decreases.This is mainly due to the fact that, with the gradual increase of size of the water pool, uidity of the medium increases.This causes the faster relaxation of the conned water molecules inside the core of reverse micelles thereby decreasing the extent of REES.So this supports our assumption that with the gradual increase of hydration, the extent of REES gradually decreases, implying that the motional restriction is gradually decreasing with the increase in water content.

Time resolved anisotropy studies
Rotational relaxation studies further helps us to understand the formation of a supramolecular host-guest complex inside the core of reverse micelle.In pure water 7-DCCA shows rotational relaxation time of $90 ps.In water solution of 7-DCCA (3 Â 10 À6 (M)) aer the addition of 7.30 mM HP-g-CD the rotational relaxation time increases to 0.684 ns.This indicates the formation of a supramolecular host guest complex between the dye 7-DCCA and macrocycle HP-g-CD.Now aer the incorporation of 7-DCCA-HP-g-CD host guest complex inside the core of reverse micelle, the average rotational relaxation time increases signicantly to 3.561 ns at w 0 ¼ 3. The rotational relaxation time gradually decreases with the gradual increase of w 0 value i.e. the size of the water pool inside the core of reverse micelle (Fig. 6 and Table 4).At w 0 ¼ 30 the average rotational relaxation time becomes 0.780 ns.The most noteworthy thing of this study is that in the case of doubly conned system at every w 0 value the rotational relaxation time is prominently higher than that in singly conned system.We have tted the anisotropy decays by biexponential function.We have found that for all the w 0 values, both the components are higher in timescale than that in singly conned system. 47This clearly indicates that aer the double connement of the dye inside the core of water/AOT/isooctane reverse micelle the rotational relaxation of the 7-DCCA decreases.This indicates the existence of the dye-macrocycle complex inside the core of reverse micelle.We have observed that even at w 0 ¼ 30 the average rotational relaxation time of 7-DCCA in the doubly conned system is higher than that in single conned system.This proof the existence of supramolecular host guest complexation inside the core of reverse micelle and this complex exist even at w 0 ¼ 30.With the gradual increase of size of the water pool, the restriction over the dyemacrocycle complex gradually decreases, so the rotational relaxation time also gradually decreases.When the size of reverse micelle is small, the frozen nature of the water pool prevents the rotational motion of the dye macrocycle complex, causing high rotational relaxation time.This relaxation time is higher than that of singly conned dye at same w 0 value.With the gradual swelling of the reverse micelle the movement of the water molecules inside the core increases and it decreases the restriction on the rotation of the dye-macrocycle complex, hence reducing the rotational relaxation time.

Conclusion
In this study we have shown the double connement of a hydrophilic dye 7-DCCA in CD/reverse micelle system, by rst encapsulating dye in HP-g-CD and then trapping the complex inside the core of the reverse micelle.The absorption spectra of 7-DCCA in doubly conned system shows signicant blue shi compared to the singly conned system in the reverse micelle.We have observed red shied emission spectra of 7-DCCA in our present system at every w 0 value than the singly conned system.The uorescence decay times showed the effect of double connement on the photophysics of 7-DCCA in the reverse micelles.Solvent relaxation dynamics shows that the dye 7-DCCA faces greater restriction in the doubly conned system than in the singly conned system in the reverse micelle.This type of double connement in the presence of supramolecular host in the water pool of reverse micelle is further substantiated by the time resolved anisotropy study.The rotational relaxation time of 7-DCCA in the doubly conned system is always higher than the singly conned system.This clearly indicates that due to the double connement of 7-DCCA inside the reverse micelle the rotational relaxation time is higher compared to the single conned system.
r 0 is the limiting anisotropy, b represents the relative contribution of the slow component and (1 À b) represents the relative contribution of the fast component.The slow and the fast components are denoted by s slow and s fast .The average rotational relaxation time is represented by the following equation: 49

Fig. 2
Fig. 2 (a) The change in steady state fluorescence emission spectra of 7-DCCA in the doubly confined system in reverse micelles with increasing w 0 value (l exi ¼ 405 nm).Inset shows the variation of fluorescence intensity with the variation of w 0 value.(b) The variation of red edge excitation shift (REES) with varying the w 0 value.

Fig. 3
Fig. 3 (a) The change in fluorescence decay time of 7-DCCA in the doubly confined system in reverse micelles with increase w 0 value (l exi ¼ 405 nm).(b) The change of ln(k nr ) value with increase in w 0 value.

Table 2
The fluorescence lifetime components of 7-DCCA in the doubly confined system in the water/AOT/isooctane reverse micelles at different w 0 values

Table 3
The decay characteristics of C(t) of 7-DCCA in the doubly confined system

Table 4
The rotational relaxation time of 7-DCCA in the doubly confined system