Unusual H-Type Aggregation of Coumarin-481 Dye in Polar Organic

Article pubs.acs.org/JPCA

Unusual H‑Type Aggregation of Coumarin-481 Dye in Polar Organic Solvents Poonam Verma† and Haridas Pal*,‡ †

Radioanalytical Chemistry Division, and ‡Radiation & Photochemistry Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India ABSTRACT: Coumarin-481 (C481) dye shows intriguing time-resolved (TR) fluorescence behavior in polar organic solvents of both protic and aprotic nature, namely, ethanol (EtOH) and acetonitrile (ACN), demonstrating the presence of multiple emitting species in the solution. Following concentration-dependent and wavelength-dependent TR fluorescence measurements and the time-resolved emission spectra (TRES) and time-resolved area-normalized emission spectra (TRANES) subsequently constructed using wavelength dependent decay parameters, we convincingly conclude that in the studied solvents a part of the dissolved dye undergoes H-type of aggregation, even at a very low dye concentration. This is quite an unusual finding because the dye C481 apparently shows reasonably good solubility in these organic solvents. As the TR measurements indicate, major contribution in the fluorescence decays is due to monomeric dye, having reasonably short lifetime (∼0.64−0.68 ns), which is in accordance with the conversion of fluorescent intramolecular charge transfer (ICT) state to nonfluorescent twisted intramolecular charge transfer (TICT) state suggested for the dye in high polarity solvents, causing an efficient nonradiative deexcitation. The minor contributions arising from the aggregated dyes show its clear presence in the decays at the blue edge of the emission spectra and have relatively longer lifetimes (∼1.2−5.2 ns) because the steric hindrance caused by the stacked dyes resists the ICT to TICT conversion. Aggregation of C481 dye as observed in the present study in polar organic solvents is an intriguing finding, as the dye is a widely used fluorescent probe for various photochemical studies, where overlooking such aggregation can mislead the observed results.

1. INTRODUCTION The 1,2-benzopyrone derivatives, commonly known as coumarin dyes, are the well-known laser dyes in the blue− green region.1−6 Among various coumarin derivatives, the ones having differently substituted 7-amino groups to the basic coumarin moiety have attracted tremendous attention in diverse research areas. These dyes, generally called as the 7aminocoumarins, show very interesting excited state properties, e.g., strong intramolecular charge transfer (ICT) character,7−11 large solvent polarity dependent shifts in absorption and fluorescence spectra,7−11 very high fluorescence quantum yields (Φf), long fluorescence lifetimes (τf),7−14 etc. These interesting properties along with their high photostability have made the 7aminocoumarin dyes as the useful fluorescence probes in investigating various chemical and physiochemical processes.15−20 Potential of 7-aminocoumarin derivatives as chemosensors has also been well studied.21−23 Some coumarin derivatives have also found prospective applications in biological and biomedical sciences.24−27 Though coumarin dyes have been extensively used in different studies and the reported literatures on the photophysics of coumarin dyes is also quite rich, yet many of the atypical behaviors of some of these dyes in different solvent environments are still the topic of intense research in photochemical sciences.7−15,28−34 In one of our recent studies, we have observed an intriguing behavior of a 7-N,Ndialkylaminocoumarin dye, namely, coumarin-481 (C481), © 2013 American Chemical Society

undergoing H-type of dimer and higher aggregate formation in aqueous solution.35 Most importantly, unlike most other cases of H-aggregates that show nonfluorescent character,36−42 the aggregated species of C481 dye are found to be quite emissive in nature.35 From the detailed analysis of the timeresolved (TR) fluorescence results, it was evident that the fluorescence lifetimes (τf) of H-dimers and H-aggregates of C481 dye are longer than that of the monomeric dye, and the results have been in accordance with the suggested additional nonradiative relaxation in higher polarity solvents involving the fluorescent ICT state to nonfluorescent twisted intramolecular charge transfer (TICT) state conversion,43−45 a process that becomes sterically restricted in the dye aggregates.35 With the intriguing results of dimerization/aggregation of C481 dye in aqueous solution, we were curious if similar aggregation is also possible for the dye in other solvents, especially in polar organic solvents. This inquisitiveness was further instigated by the results on some of the coumarin derivatives showing aggregation and/or self-assembly formation in the environments like silica/methanol interfaces,46 mesoporous molecular sieves,47 alcoholic solvents and their mixtures,48 or during solvent-exchange process.49 Though photophysical behavior of C481 dye in different protic and Received: September 20, 2013 Revised: October 28, 2013 Published: October 29, 2013 12409

dx.doi.org/10.1021/jp409405a | J. Phys. Chem. A 2013, 117, 12409−12418

The Journal of Physical Chemistry A

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respectively, and used without further purification. All the present measurements were carried out at ambient temperature (25 ± 1 °C). Absorption spectra were recorded using a JASCO UV−vis spectrophotometer, model V-650 (Tokyo, Japan). Steady-state (SS) fluorescence spectra were recorded using a Hitachi spectrofluorimeter, model F-4500 (Tokyo, Japan). Timeresolved (TR) fluorescence was measured using a timecorrelated single photon counting (TCSPC)53,54 spectrometer from Horiba Jobin Yvon IBH, U.K., where samples were excited with a 374 nm diode laser (repetition rate 1 MHz, pulse width ∼100 ps), and fluorescence decay was collected at right angle geometry using a MCP PMT (IBH, Scotland, U.K.). All the measurements were carried out with analyzer at magic angle polarization with respect to the vertically polarized excitation beam to avoid any rotational depolarization effect of the dye on the observed fluorescence decays. Measured decays were analyzed using a DAS-6 reconvolution analysis software obtained from IBH. The instrument response function (IRF) for the experimental setup was recorded by replacing the sample cell with a dilute scatterer solution (suspended TiO2 particles in water) and used in the reconvolution analysis. A typical IRF for the present setup is ∼110 ps at full width at halfmaximum (fwhm), and the shortest lifetime measurable using reconvolution analysis is about 30 ps.53,54 Observed decays were fitted in general as a sum of exponentials

aprotic organic solvents of various polarities have been reported earlier and the results have been interpreted exclusively on the basis of monomeric dyes in the solution,14,29,43−45,50−52 a revisiting on the behavior of the dye in the conventional organic solvents seems essential, anticipating a possible aggregation of the dye in some of these solvents. This is far more important for the present dye because in higher polarity solvents the excited C481 dye undergoes an efficient ICT to TICT conversion, causing a large reduction in its fluorescence quantum yield and lifetime.14,29,43−45,50−52 Thus, under this situation, even a small extent of dye aggregation can appreciably affect the observed results, though in the lower polarity solvents the effect could be of less importance due to the absence of ICT to TICT conversion process, which causes a very high Φf and long τf values of the dye.14,29,43−45,50−52 Exploration on the possible aggregation of C481 dye in conventional polar organic solvents is also important form the viewpoint that the dye along with other 7-aminocoumarin derivatives are being extensively used as the extrinsic fluorescent probes in perceiving mechanism and dynamics of various physicochemical processes15−30 where dye aggregation can adversely affect the observed results. With this perspective, in the present study, we have systematically investigated the photophysical properties of C481 dye in two conventional polar organic solvents, namely, polar protic solvent ethanol (EtOH) and polar aprotic solvent acetonitrile (ACN), to understand the possible aggregation of the dye in these solutions. The chemical structure of the studied dye is shown in Chart 1 for a quick visualization.

I (t ) =

∑ Bi exp(−t /τi)

(1)

where τi is the fluorescence lifetime and Bi is the preexponential factor for the ith component of the decay. The quality of the fits were judged from the reduced chi-square (χ2) values and the distribution of the weighted residuals among the data channels.53,54 The percentage contributions (ai) of the individual lifetime components (τi) in the multiexponential decays were calculated by using the following relationship:

Chart 1. Chemical Structure of Coumarin-481 Dye

a i = {Bi τi /(∑ Bi τi)} × 100

2. MATERIALS AND METHODS Laser grade C481 sample was obtained from Exciton, USA, and used as received. Spectroscopic grade EtOH and ACN were obtained from Les Alcools de Commerce Inc. (Brampton, Ontario, Canada) and S D Fine Chem Ltd. (Mumbai, India),

(2)

3. RESULTS AND DISCUSSION 3.1. Ground State Absorption and Steady-State Fluorescence Measurements. Ground state absorption spectra of C481 dye in EtOH and ACN solutions were

Figure 1. (A) Absorption spectra of C481 dye in EtOH solutions. Dye concentrations for the spectra 1−7 are 35.0, 17.2, 9.6, 5.3, 2.6, 1.2, and 0.6 μM, respectively. (B) Absorption spectra of C481 dye in ACN solutions. Dye concentrations for the spectra 1−7 are 30.2, 15.6, 7.9, 4.1, 2.1, 1.1, and 0.6 μM, respectively. Insets of both the panels show the peak normalized absorption spectra for the highest and lowest dye concentrations used in the respective solvents. 12410



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Figure 2. (A) Fluorescence spectra of C481 dye in EtOH solution. Dye concentrations for the spectra 1−6 are 9.6, 5.3, 2.6, 1.2, 0.6, and 0.3 μM, respectively. (B) Fluorescence spectra of C481 dye in ACN solutions. Dye concentrations for the spectra 1−6 are 15.6, 7.9, 4.1, 2.1, 1.1, and 0.6 μM, respectively. Insets of both the panels show the peak normalized fluorescence spectra for the highest and the lowest dye concentrations in the respective solvents. Excitation wavelength was 374 nm.

Figure 3. (A) Peak-normalized fluorescence spectra of C481 dye (16.8 μM) in EtOH; excitation wavelengths 450 nm (black), 404 nm (red), 374 nm (blue), 350 nm (green), and 340 nm (magenta). (B) Peak-normalized fluorescence spectra of C481 dye (15.5 μM) in ACN; excitation wavelengths 450 nm (black), 399 nm (red), 374 nm (blue), 350 nm (green), and 340 nm (magenta).

Figure 4. Peak-normalized fluorescence excitation spectra of C481 dye in (A) EtOH and (B) ACN solutions. The emission wavelengths were 480 nm (blue end; blue), 514 nm (peak; green), 570 nm (red end; red) in EtOH and 450 nm (blue end; blue), 504 nm (peak; green), 570 nm (red end; red) in ACN, respectively. Panels I and II for each of the solvent systems correspond to the dye concentrations of 2.7 and 16.8 μM in EtOH and 3.1 and 15.5 μM in ACN solutions, respectively.

more evidently seen by plotting the peak normalized absorption spectra, shown in the insets of Figure 1A,B in the respective solvents, for the highest and the lowest concentration of the dye used. Like absorption spectra, the steady-state (SS)

measured at different dye concentrations and are shown in Figure 1A and B, respectively. As indicated from these figures, there is no obvious change in the spectral characteristics of the dye on changing the dye concentration in the solutions. This is 12411



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Figure 5. Fluorescence decays of C481 dye in EtOH solution; (A) measured at the emission peak (514 nm) and (B) measured at a blue-shifted emission wavelength (480 nm). Dye concentrations for the decays shown in black, red, blue, magenta, green, and violet are 9.68, 4.84, 2.24, 1.21, 0.61, and 0.30 μM, respectively. Excitation was done at 374 nm.

Figure 6. Fluorescence decays of C481 dye in ACN solution; (A) measured at the emission peak (504 nm) and (B) measured at a blue-shifted emission wavelength (450 nm). Dye concentrations for the decays shown in black, red, blue, magenta, green, and violet are 15.0, 7.8, 3.8, 1.9, 0.94, and 0.47 μM, respectively. Excitation was done at 374 nm.

fluorescence spectra, shown in Figure 2A,B for EtOH and ACN solutions, respectively, also do not indicate any obvious change in the spectral characteristics on changing the dye concentrations. Comparison of the peak normalized fluorescence spectra for the highest and the lowest dye concentration in the two respective solvents are shown in the insets of Figure 2A,B, to better envisage the above fact. Thus, on the basis of the ground state absorption and SS fluorescence results, it is indicated that the dye C481 apparently behave quite normally both in the polar protic (EtOH) and polar aprotic (ACN) solvents, though these measurements are not sensitive enough to identify a small extent of abnormality in the behavior, and thus, further sensitive measurements are required to unequivocally establish the exact characteristics of the dye in these solvent systems. To investigate the present systems further, we also recorded the SS fluorescence spectra of the dye in both EtOH and ACN solutions as a function of the excitation wavelength, covering both the blue edge and the red edge of the absorption band. The results thus obtained in the two solvents are shown in Figure 3A and B, respectively. It is indicated from the observed results that the fluorescence spectra of the dye are effectively independent of the excitation wavelength used. These results thus corroborate well with the other results shown in Figures 1 and 2 and seemingly indicate the predominant existence of the monomeric form of dye in both EtOH and ACN solutions, if not exclusively. In the present context, we also recorded the excitation spectra of the dye keeping the emission wavelengths at extremely blue edge, at the peak, and at the extremely red edge of the emission spectra. These results in EtOH and ACN

solutions are shown in Figure 4A and B, respectively. Like the emission results in Figure 3A,B, the excitation spectra in Figure 4A,B also do not show any significant changes with the changing emission wavelengths. Since SS fluorescence and ground state absorption measurements are quite less sensitive techniques, to identify any small changes in the characteristics of the present dye in the studied solvent systems, we resorted to the more sensitive time-resolved (TR) fluorescence measurements, as are discussed in the next section. 3.2. Time-Resolved Fluorescence Measurements. Though literature reports on the photophysical properties of 7-aminocoumarin dyes in conventional organic solvents explicitly indicate nothing about their aggregation,29,43−45,51,52 yet our recent results on C481 dye in aqueous solution showing significant H-dimer/H-aggregate formation35 and the reports of the self-assembly formation and/or aggregation of some of the coumarin derivatives under environments like solid-solvent interfaces, molecular sieves, alcoholic solvents, their mixtures, etc.,46−49 incited our inquisitiveness to look into the possibility of any such aggregation of C481 dye in both polar protic and polar aprotic organic solvents (like EtOH and ACN). Since TR fluorescence is a very sensitive technique to detect multiple emissive species,53−55 in the present study we carried out such measurements, recording the fluorescence decays of the dye in both EtOH and ACN solutions, both as a function of the changing dye concentration and the changing monitoring emission wavelength. Fluorescence decays of C481 dye in EtOH and ACN solutions measured at the emission peak (514 nm in EtOH and 504 nm in ACN) and at a significantly blue-shifted emission 12412



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relatively weaker emissions and shorter fluorescence lifetimes for these species than the dimers. The net result would thus be an apparent increase in the decay tail with decreasing dye concentration. Present proposition is further supported by the fact that the decay tail gradually becomes more extended as the dye concentration decreases, indicating an increasing contribution of the longer lifetime dimeric emissive species. To be mentioned here, a very similar observation was also made in our earlier study on C481 dye in aqueous solution on reducing the dye concentration, and the results were interpreted in terms of the changing relative contributions of the dimers and higher aggregates.35 Analysis of the concentration-dependent decays in Figures 5 and 6 in general required biexponential function to obtain acceptable fits (χ2 values around 1.0 and weighted residuals distributed randomly among data channels).53−55 The decay parameters thus obtained at different dye concentrations and monitoring emission wavelengths in EtOH and ACN solutions are listed in Tables 1 and 2, respectively. To be mentioned, that

wavelength (480 nm for EtOH and λem = 450 nm for ACN) as a function of the dye concentration are shown in Figures 5 and 6, respectively. In EtOH solution, though the decays at the emission peak (cf. Figure 5A) do not show any drastic change, yet the presence of a small extent of decay tail whose contribution changes with the dye concentration clearly suggests the existence of at least two emissive species in the solution. This effect is more pronouncedly observed in the decays measured at the blue edge of the emission spectra, shown Figure 5B. For ACN solution, the nature of the decays is more or less similar to that in EtOH solution, though in this case the tailing is not that elongated as in EtOH. To be mentioned, a similar kind of tailing in the fluorescence decays of C481 dye was also observed in aqueous solution and rationalized on the basis of aggregation of the dye in the solution.35 Drawing an analogy with the results for C481 dye in aqueous solution, tailing of the fluorescence decays in the present organic solvents is also attributed to a small extent of aggregation of the dye in the polar organic solvents like EtOH and ACN. Obviously, the major contribution in the decays with an apparently shorter decay-time (τ1) is due to the monomers of the dye. Present assertions are based on the consideration that in high polarity EtOH and ACN solvents the dye monomers would undergo efficient ICT to TICT conversion in the excited state, causing the fluorescence lifetime to be significantly shorter.43−45,50−52 In the dye aggregates, however, the ICT to TICT conversion process would be largely restricted due to steric constraints of the stacked dyes, causing the lifetime (τ2) of the aggregated species to be longer than that of the dye monomers.35 From the observed decays in Figures 5 and 6, it is very evident that the contribution of the decay tails is always larger at the blue-shifted emission wavelengths than at the emission peak. Such an observation suggests that, like in aqueous solution reported earlier,35 the aggregation of C481 dye in EtOH and ACN solutions also results in the formation of Htype dimers/aggregates. Important to mention here is that even though in most cases the H-aggregates are known to be nonemissive in nature, there are cases where reasonably strong emissive H-aggregates are observed for some of the chromophoric dyes.35,42,48 In fact, in a recent report, Cigan and co-workers48 have shown the formation of highly emissive H-dimers for a 7-aminocoumarin derivative in polar protic organic solvents like EtOH and MeOH, and the authors suggest that the aggregation in these cases is mainly driven by strong π+−π− interaction that helps in overcoming the restriction imposed on the electronic transition in the aggregates arising from Kasha’s exciton theory based on simple van der Waals interactions. It is expected that the emissive nature of the H-dimers/H-aggregates for C481 dye in EtOH and ACN solutions is also due to similar π+−π− interaction as suggested by Cigan and co-workers. Another important point to be noted in the present context is that the contribution of the aggregate emission appearing as the decay tails gradually increases with a decrease in the dye concentration. This observation seems apparently unusual because aggregation is supposed to be more pronounced on increasing the dye concentration. We feel that the decrease in the dye concentration leads to an increase in the H-dimer/ higher dye aggregates ratio, which causes an apparent increase in the decay tail. It is expected that because of the participation of the exciton−exciton annihilation mechanism,56−58 the larger aggregates will suffer a much faster excitation decay, causing

Table 1. Fluorescence Decay Parameters for C481 Dye in EtOH Solution As Estimated at Different Dye Concentrations and at Different Monitoring Emission Wavelengths; Excitation Wavelength in All the Cases Was λex = 374 nm λem = 480 nm [C481] (μM)

a1 (%)

35.0 17.2 9.6 5.3 2.6 1.2 0.6

99.2 99.1 99.0 98.9 98.9 98.2 96.3

[C481] (μM)

a1 (%)

35.0 17.2 9.6 5.3 2.6 1.2 0.6

99.6 99.6 98.5 99.8 99.8 99.6 98.8

τ1 (ns)

τ2 (ns)

χ2

0.8 0.9 1.0 1.1 1.1 1.8 3.7

1.22 1.30 2.30 2.63 4.09 4.39 5.12

1.01 1.10 1.15 1.01 1.16 1.19 1.01

τ1 (ns)

a2 (%)

τ2 (ns)

χ2

0.63 0.62 0.64 0.64 0.66 0.64 0.64

0.36 0.39 0.54 0.21 0.22 0.44 1.22

1.19 1.37 2.16 2.59 4.03 4.32 5.12

1.00 1.02 1.06 1.10 0.97 0.98 1.00

a2 (%)

0.62 0.63 0.64 0.63 0.64 0.64 0.64 λem = 514 nm

in ACN solution, though the decays at the emission peak could be fitted reasonably well following a single-exponential function, yet to keep a homogeneity with the other clearly biexponential decays, a biexponential analysis (cf. Table 2) was also carried out for the former decays, and thus, suitable correlations were obtained among different decay parameters with changing dye concentration and monitoring emission wavelengths. As are seen from Tables 1 and 2, in both the solvents, the decays are always dominated by shorter decay component, τ1, having a lifetime value of about 0.64 ns in EtOH and about 0.67 ns in ACN. This component in both the solvents is effectively independent of the dye concentration and the monitoring emission wavelength. Expecting that the neutral dye C481 would preferentially exist in its monomeric form in an organic solvent, and considering that the high polarity of EtOH and ACN would assist ICT to TICT conversion for the excited 12413



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In addition to a shorter τ1 component, the fluorescence decays also contain a relatively longer component τ2 in both EtOH and ACN solutions (cf. Tables 1 and 2). We suppose that this τ2 component is due to the dye aggregates present in the solution. As one would notice from Tables 1 and 2, the contribution of τ2 is higher at blue-shifted emission wavelength than at the emission peak, which is in accordance with the Htype of aggregation of the dye in the present solvent systems, as was also observed earlier in aqueous solution.35 In EtOH solution, it is seen that time constant for the τ2 component gradually increases on decreasing the dye concentration. Such an observation is indicative of the presence of more than one type of aggregate in the solution. Considering that in the aggregated dyes the ICT to TICT conversion would be sterically hindered and that the exciton−exciton annihilation would be less effective in the dimers than in the higher aggregates, we feel that the increased τ2 value with decreasing dye concentration is due to gradual conversion of higher aggregates to dye dimers. Accordingly, the longest lifetime value of ∼5.12 ns, as estimated for the most dilute solution in EtOH could most likely be closer to the lifetime of the H-dimer in the solution. At higher dye concentrations, the estimated τ2 values, however, might not represent any particular dye aggregate but would be due to overall contributions from different dye aggregates (dimer, trimer, tertramer, etc.) in the solution. Unlike in EtOH solution, in ACN solution, the τ2 value does not show any noticeable change with the changing dye concentration, though the contribution of this component clearly increases as the dye concentration is reduced (cf. Table 2). Expecting that the dye dimers would not have largely different lifetimes in EtOH and ACN solutions, we presume that in ACN solution the decay tails are mostly contributed by the dye aggregates higher than dimers. Thus, the results indicate that, while the dimers are quite preferred in polar protic solvent EtOH, the higher dye aggregates are mostly preferred in polar aprotic solvent ACN. This proposition is also substantiated by the observation that the τ2 values estimated in ACN solution are quite closer to the τ2 values in EtOH solution at the higher dye concentrations, where larger aggregates would dominate. Assuming the above situations prevailed in the two solvents, it is imperative to infer that, while the higher solvent polarity drives the aggregation of C481 dye, the protic nature of the solvent help in the stabilization of the dimeric species. The

Table 2. Fluorescence Decay Parameters for C481 Dye in ACN Solution As Estimated at Different Dye Concentrations and at Different Monitoring Emission Wavelengths; Excitation Wavelength in All the Cases Was λex = 374 nm λem = 450 nm [C481] (μM)

a1 (%)

30.2 15.6 7.9 4.1 2.1 1.1

92.2 91.7 92.4 91.9 91.0 89.4

τ1 (ns)

a2 (%)

0.66 7.8 0.66 8.3 0.68 7.6 0.66 8.1 0.67 9.0 0.66 10.6 λem = 504 nma

τ2 (ns)

χ2

1.64 1.61 1.67 1.72 1.71 1.64

1.18 1.00 1.08 0.99 1.01 1.10

[C481] (μM)

a1 (%)

τ1 (ns)

a2 (%)

τ2 (ns)

30.2

98.5 (100)

0.67 (0.67)

15.6

98.7 (100)

0.67 (0.69)

7.9

98.9 (100)

0.67 (0.68)

4.1

98.8 (100)

0.68 (0.68)

2.1

99.0 (100)

0.68 (0.67)

1.1

98.7 (100)

0.66 (0.68)

1.5 (---) 1.3 (---) 1.1 (---) 1.2 (---) 1.0 (---) 1.3 (---)

1.66 (---) 1.66 (---) 1.65 (---) 1.66 (---) 1.65 (---) 1.65 (---)

χ2 0.98 (1.08) 1.01 (1.13) 1.00 (1.10) 0.97 (1.06) 0.98 (1.10) 1.01 (1.18)

a

The decays at the emission peak of the dye in ACN could also be fitted reasonably well following single-exponential function, and the decay parameters thus obtained are listed in the parentheses.

dye,43−45,50−52 the shorter τ1 component is attributed to the monomeric dyes present in the solution. To be noted that in the literature the fluorescence lifetime of C481 dye in EtOH and ACN solutions are reported to be in the range of about 0.6−0.9 and 0.6−0.7 ns, respectively, quite similar to the τ1 values estimated in the present study. As reported in the literature, unlike in high polarity solvents, the lifetime values for C481 dye in nonpolar to moderately polar organic solvents are quite long, in the range of about 4−6 ns. Such longer lifetime is due to the absence of ICT to TICT conversion, as the lower solvent polarity cannot stabilize the highly polar TICT state.43−45,50−52

Figure 7. (A) Fluorescence decays of C481 dye (2.70 μM) in EtOH solution measured at different emission wavelengths of the dye as indicated in the figure. (B) Fluorescence decays of C481 dye (2.67 μM) in ACN solution measured at different emission wavelengths as indicated in the figure. In both the cases the decays show the appearance of extended tails at the shorter wavelengths. Excitation was done at 374 nm. 12414



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Figure 8. (A) Time-resolved emission spectra (TRES) and (B) time-resolved area normalized emission spectra (TRANES) of C481 dye (2.7 μM) in EtOH solution. The time snaps for the spectra with decreasing intensity and blue shift are 0.05, 0.1, 0.15, 0.2, 0.3, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0, and 10.0 ns, respectively. The inset of panel A shows the changes in the emission maxima of the TRES and TRANES with time.

Figure 9. (A) Time-resolved emission spectra (TRES) and (B) time-resolved area normalized emission spectra (TRANES) of C481 dye (2.67 μM) in ACN solution. The time snaps for the spectra with decreasing intensity and blue shift are 0.05, 0.1, 0.2, 0.3, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0, and 10.0 ns, respectively. The inset panel A shows the changes in the emission maxima of the TRES and TRANES with time.

fact that the protic nature of the solvents provides an additional preference for the formation of C481 dimers is also supported from our earlier results on C481 dye in aqueous solution,35 where dimeric contributions were found to be much more pronounced than even in EtOH solution. 3.3. Time-Resolved Emission Spectra of C481 Dye in EtOH and ACN Solutions. Wavelength dependent fluorescence decays covering the whole SS emission spectra of C481 dye were measured in both EtOH and ACN solutions to construct time-resolved emission spectra (TRES),59,60 which were further converted to time-resolved area normalized emission spectra (TRANES)61,62 of the dye in the respective solvents to understand the time-dependent changes in spectral features of different emitting species. As required in different cases, the observed decays were analyzed, using either mono-, di-, or triexponential functions. Using the estimated decay parameters at different wavelengths, the TRES and TRANES were constructed using the standard procedure.59−62 Necessary details of the TRES and TRANES construction are already given in our earlier paper.35 Figure 7A,B shows the typical wavelength-dependent decays for C481 dye in EtOH and ACN solutions, respectively. Clearly, at shorter wavelengths the decays are nonsingle exponential with quite increased contribution of the extended decay tails, which gradually decreases on moving to longer wavelengths. This feature of the wavelength-dependent decays is consistent with the suggested H-type of aggregation of the dye in the present solvent systems.

The TRES and TRANES for C481 dye constructed in EtOH solution are shown in Figure 8A and B, respectively. As indicated from Figure 8A, fluorescence intensity decreases very sharply for the initial time span of about 3 ns. During this time, however, there is hardly any shift in the spectral position, as clearly indicated from Figure 8B and the inset of Figure 8A. These observations are in accordance with our inference that the major contribution in the fluorescence decays is due to excited dye monomers having a reasonably short fluorescence lifetime of ∼0.64 ns. At the latter time span, beyond about 3 ns, there is a significant blue shift in the TRES and TRANES, as evident from Figure 8B and the inset of Figure 8A. This observation evidently indicates that the major emitting species at the latter time span have largely blue-shifted emission spectra in comparison to that of the monomeric emission. Assuming the longer lifetime components measured in EtOH solution at different dye concentrations (cf. τ2 in Table 1) are due to the emissions from the dye aggregates, the blue shift in the TRES and TRANES clearly suggests that the dye C481 undergoes Htype of aggregation in the present solvent system because the alternative J-type of aggregation should always lead to largely red-shifted emission.36−42 To be mentioned here, though blue shift in the absorption spectra is a characteristic feature for the H-type aggregates, the emission spectra of these aggregates can possibly exhibit either a blue shift or a red shift, depending on whether the emission arises from the higher or the lower excitonic states. To be mentioned, however, in most cases, the H-aggregates are observed to be either very weakly fluorescent 12415



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restriction toward ICT to TICT conversion, caused by the steric hindrance imposed by the stacked dyes. Comparison of the results in EtOH and ACN solutions suggests that, while the polar protic solvent EtOH supports dimer formation better, the polar aprotic solvent ACN mostly leads to the formation of higher aggregates than dimers. Since the TRES and TRANES show large blue shift at longer times (beyond ∼3 ns) and the fluorescence decays show increasingly more aggregate contributions on moving toward shorter emission wavelengths, we convincingly conclude that in the present solvent systems the dye undergoes an H-type aggregate formation, which was also observed earlier in aqueous solution, albeit with a much more dominating aggregation process.35 Aggregation of C481 dye as indicated in this study in polar organic solvents is not only an unusual observation but also an extremely important finding because the dye C481 is a well utilized fluorescence probe for various photochemical studies where such dye aggregation can adversely modulate the observed results and hence influence their interpretations.

or completely nonfluorescent in nature, mainly because for their unusually fast nonradiative deexcitation rates.36−42 The fact that the H-type aggregates for C481 dye show a blue shift in the emission spectra in EtOH and ACN solutions, as also observed earlier in aqueous solution,35 suggests that the emission in the present cases arises mainly from the higher excitonic states of these aggregates. Important to note here is that a similar kind of blue-shifted emission from H-aggregates of a 7-aminocoumarin dye has also been observed by Cigan and co-workers in alcoholic solvents.48 Thus, blue-shifted emission from H-aggregates seems not to be very unusual, especially for the 7-aminocoumarin derivatives, though emission yields of these aggregates are certainly very low. The TRES and TRANES for C481 dye constructed in ACN solution are shown in Figure 9A and B, respectively. As indicated from these figures, the major features in the TRES and TRANES for the dye in ACN solution are very similar to those in EtOH solution. Thus, there is a sharp decrease in the fluorescence intensity at the initial time span of about 3 ns and a significant blue shift in the emission spectra in a latter time span, beyond about 3 ns. Like EtOH solution, the observations in ACN solution are also interpreted as due to dominant monomeric emission at the initial times and dominant aggregate emissions at the latter times. Moreover, the blue shift in the spectra at the longer times also supports that, like in EtOH solution, the dye also undergoes H-type of aggregate formation in polar ACN solution. Overall, the observations in the TRES and TRANES are in good agreement with the proposed aggregation of the dye in the polar organic solvents studied. Present results indicate that even in the submicromolar concentration range there is quite reasonable aggregation of the dye in the studied polar organic solvents. This is an intriguing observation considering the fact that C481 dye along with other aminocoumarins is extensively used as a fluorescence probe in studying various physicochemical processes where dye aggregation can adversely modulate the observed results and their interpretations.

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AUTHOR INFORMATION

Corresponding Author

*(H.P.) E-mail: [email protected]. Fax: 022-25505151 and 02225519613. Tel: 022-25595396. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are thankful to Dr. B. S. Tomar, Head, RACD, Dr. D. K. Palit, Head, RPCD, Dr. K. L. Ramakumar, Director, Radiochemistry & Isotope Group, and Dr. B. N. Jagatap, Director, Chemistry Group, for their constant encouragement and support in our research endeavors.

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REFERENCES

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4. CONCLUSIONS Photophysical properties of coumarin-481 (C481) dye shows unexpected aggregation in polar protic as well as in polar aprotic organic solvents, namely, ethanol (EtOH) and acetonitrile (ACN), respectively, even though the dye solubility in these solvents are apparently quite high. Albeit the aggregation is not as severe as in aqueous solution reported earlier,35 the effect is quite substantial to affect the fluorescence decays, which clearly indicate the presence of multiple emitting species in the solution. As indicated from concentration- and wavelength-dependent TR measurements, the major contribution in the decays arises from monomeric dye, showing lifetimes (τ1) of ∼0.64 and ∼0.68 ns in EtOH and ACN solvents, respectively. Such short fluorescence lifetimes for the dye monomers is due to efficient ICT to TICT conversion, supported by high polarity of the studied solvents. A minor contribution in the decays appearing as the tails is due to aggregated dyes, showing concentration-dependent changes in the lifetime (τ2) values between ∼1.2 to ∼5.2 ns in EtOH and almost a constant lifetime of ∼1.7 ns in ACN, ascribed due to varying stabilization of the dimers in the protic and aprotic solvents. Accordingly, the τ2 value of ∼5.2 ns at very low dye concentration in EtOH solution is suggested to be closer to the lifetime of the dimeric dye in the solution. Relatively longer lifetimes for the dye aggregates than monomers are due to 12416



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Unusual H-Type Aggregation of Coumarin-481 Dye in Polar Organic

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