CHAPTER II EVALUATION OF GROUND AND EXCITED STATE DIPOLE MOMENTS

Transcription

1 CHAPTER II EVALUATION OF GROUND AND EXCITED STATE DIPOLE MOMENTS

2 III.1. INTRODUCTION The nature and the energy of electronically excited state of a dye molecule determine its photophysical properties. In quantum calculations, the molecule is assumed absolutely to be isolated, as if it were in the gas phase at very low pressure. Since we are concerned with molecules in condensed phases, liquids, viscous liquids and solids, the effect of electronic states of solute molecules, is therefore, of substantial importance in photophysics [1] and photochemistry [2]. A molecule has a number of electronic states, which are defined by their energy E, and wavefunctionψ, according to Shrödinger equation E ψ = Hψ. The wavefunction signifies the nature of the state, as different from its energy; it describes, in particular, the electron distribution within the molecule, which is closely related to its chemical reactivity. In quantum calculations, these states have the most stable nuclear configuration (molecular geometry, i.e., bond length and bond angles) for the relevant electron distribution. Transitions between such states cannot be observed since the heavy nuclei take a long time to rearrange to their most stable geometry after the rapid change in the electron distribution; the nuclear configuration of the initial state is preserved in the Frank-Condon (FC) state reached directly after the transition (in time <10-16 s) [3]. A radiative transition (absorption or emission) connects a relaxed initial state to a FC final state. When the molecule is surrounded by a liquid solvent or solid matrix (the medium) each state is stabilized (or destabilized, as the case maybe) by an energy E s, known as the solvation energy. The difference in the solvation energies of the final and initial states in a various solvents is referred to as solvatochromic shift and is the main experimental evidence for this energy difference. Therefore, the absorption and emission spectra are more 33

3 informative in this respect as they are related to the energy of relaxed excited state. These experimental observations with solvent dependence of the energy of excited states gives rise to various theories of solvatochromic shifts and try to narrate the electronic distribution of the molecule in its excited state, especially to the total dipole moment (µ) and/or to the average polarizability (α). Determination of the ground and exited-state dipole moments of the molecules is imperative, since the values of dipole moments provide information about the change in the electronic charge distribution upon excitation. The simple physical meaning of the dipole moment allowed application to a variety of structural, in particular, stereochemical-problem, even at the early stage of its development. There are various techniques of determining excited state dipole moment µ e with electro optical methods like electrochromism of absorption and fluorescent bands [4], Stark splitting of rotational levels of the 0-0 vibrational bands and the effect of an external electric field on the fluorescence anisotropy [5, 6], nevertheless the solventshift method based on the analysis of salvatochromism of absorption and fluorescence maxima is the simplest and the most widely used. Though these methods are considered to be more accurate, they are restricted only to relatively simple molecules. In order to study the changes in electronic distribution in excited states of solute molecules, the solvent-induced shifts of electronic bands of molecules has been used extensively [7]. It is known that the loss of energy of an excited molecule in a solution, both by radiative and non-radiative processes characteristically depend upon the molecular structure and the environment of the molecules. Therefore, not 34

4 only the structure of solute molecules but the solvent also plays an important role in determining its radiative and non-radiative properties. The strong emission of dyes results from the polar character of low-lying excited states. It is identified that the electronic spectra of these molecules are affected by their milieu. The solvent effects are of particular importance, among the principal environmental factors affecting the electronic spectra. The dipole moments of a molecule in the ground state and an excited state depend on the electron distribution in these states. A change of solvent when it produces alteration in both the excitation and fluorescence wavelength indicates that the solvent, in some way, is able to interact with the solute in the ground state [8]. However, when solvent produces a shift in fluorescence wavelength alone, it indicates an interaction between the solvent and the solute molecule in the excited state. Also, when a molecule is excited, its dipole moment changes and it no more remains in equilibrium with its immediate environment. As the molecule relaxes and tries to attain equilibrium with its surroundings, some energy is dissipated in the form of non-radiative energy and the fluorescence emission wavelength gets shifted according to Frank-Condon principle [9]. In addition to this, sometimes large-shifts are produced by specific short-range interactions, e.g., hydrogen-bonding, complex formation, etc. A systematic solvent effect analysis is therefore, useful to understand the effect of the environment and various mechanisms of de-excitation and relaxation of an excited molecule in solution. These properties have been studied as a function of concentration of the dye, viscosity, polarity and polarizability of the solvents, as well as the ph 35

5 of the solution. The solvent shifts can be accounted in terms of the overall effect of the interaction forces (which are mainly of van der Waals type) on the π-electron system of the molecule. It is also known that as the π-electron system becomes less localized, the transition energy becomes smaller resulting in a bathochromic shift (red shift) and its opposite effect gives rise to a hypsochromic shift (blue shift). In order to assign the electronic transitions as π π * or n π * the solvatochromic technique is found quite informative. It is known that π π * bands show a red shift in the solvents of increasing polarity while n π * bands show a blue shift. Thus a change of solvent affects the ground and excited states differently because of the difference in the solvation energies. It may be noted that the solute-solvent interaction affects the fluorescence spectra in several ways viz., shift in the fluorescence spectrum, quenching of fluorescence by intersystem crossing (ISC) and formation of nonfluorescent complex or by ionization to non-fluorescent species. In the present study our main aim is to obtain ground and excited state dipole moments using different experimental techniques viz., solvatochromic shift method and theoretically by density functional theory (DFT) using Gaussian03 program. For this purpose, absorption and fluorescence characteristics of two dipolar fluorescent dyes namely, 2-(2, 7-dichloro-6- hydroxy-3-oxo-3h-xanthen-9-yl) benzoic acid (Fluorescein 27) and N-[6- diethylamino)-9-(2,4-disulfophenyl)-3h-xanthen-3-ylidene]-n-ethylhydroxid (Sulfarhodamine B) in various solvents have been undertaken in the present study. The molecular structures of these are shown in Fig.3.1. An 36

6 understanding of excited state properties of fluorescent dyes helps not only in the design of new molecules but also for the optimum performance for specific applications. The spectroscopic properties of fluorescein dyes are well known with the dyes having applications ranging from dye lasers [10] to tracers in flow visualization and mixing studies [11-14]. Fluorescein dyes are very attractive due to their low cost, non-toxicity, low-staining potential, high quantum yield (φ > 0.9), water-solubility, and easy optical accessibility with commercial lasers. On the other hand, the SRB has been used to measure druginduced cytotoxicity and cell proliferation for large-scale drug-screening applications [15]. The solvatochromic shifts have also been used for the determination of the excited state dipole moments of some polar dyes and coumarins [16-23]. In the following section we present the two different theoretical methods used to evaluate the ground and excited state dipole moments followed by their results and discussion. III.2. III.2.1. THEORETICAL BACKGROUND Solvatochromic shift method The expression most frequently used in fluorescence spectroscopy is, however, the somewhat simplified equation, first developed by Lippert [24, 25] and Mataga [26, 27]. It is based on the Onsager s reaction field theory, which assumes that the fluorophore is a point dipole residing in the centre of a spherical cavity with radius ( a ) in a homogeneous and isotropic dielectric with relative permitivity (ε). The so called Lippert-Mataga equation is no longer applicable when, in addition to the non-specific interactions, specific fluorophore/solvent interactions such as hydrogen bonding or electron-pair 37

7 donor/electron-pair acceptor interactions also contribute significantly to the overall solute-solvent interaction. A further limitation results from the cavity radius, which is difficult to estimate for elongated molecules with an ellipsoidal shape [28]. For quantitative assessment of the solvent-solute interactions, multiparameter solvent polarity scale and spectroscopic shifts can be used. Effect of solvent polarity on the spectral aspects of the solute can be interpreted by means of linear solvation energy relationship (LSER) concept that can be formulated as Kamlet-Abbound-Taft and Katritzky Equations (eqns.3.1 and 3.2 respectively) [29, 30]. (ν a -ν f ) = (ν a -ν f ) 0 + α +b β +s π* (3.1) (ν a -ν f ) = (ν a -ν f ) 0 + ε 1 2ε n 1 +b 2 2n + 1 +s E T (30) (3.2) where π* is a measure of the solvent polarity/polarizability [31], α is the scale of the solvent hydrogen solvent bond donor (HBD) acidities [32], β is the scale of the solvent hydrogen solvent bond acceptor (HBA) basicities [33]. ε, n and E T (30) are relative permittivity, refractive index and empirical polarity parameter of the solvent, respectively. Eqn. (3.2) estimates independent contributions of solvent dipolarity, polarizibility, and other specific interactions like hydrogen bonding and extra π π interaction. All the parameters should be re-normalized and re-scaled for Eqn. (3.2), to have comparable, b and s values. (ν a -ν f ) 0 is the regression value of the solute property in the reference solvent. The regression coefficients, b and s in these equations measure the 38

8 relative susceptibilities of the solute property, such as absorption, fluorescence and other spectroscopic parameters. Kawski [34-36] obtained a simple quantum mechanical second order perturbation theory of absorption (ν a ) and fluorescence (ν f ) band shifts in different solvents of varying permitivity (ε) and refractive index (n) relative to the band position of a solute molecule, based on which the following equations are obtained: ν a -ν f =m 1 f (ε, n) + const. (3.3) ν a +ν f =-m 2 [ f (ε, n) + 2g(n)] + const. (3.4) where f 2 2 2n + 1 ε 1 n 1 ε, n) = (3.5) 2 n + 2 ε + 2 n + 2 ( 2 is the polarity of the solvent [37] and with g(n)= 4 3 n ( n + 2) µ µ e g m = 1 hca 3 (3.6) (3.7) and m µ µ e g = hca 3 (3.8) h being Planck s constant and c, the velocity of light in vacuum. The parameters m 1 and m 2 can be determined from absorption and fluorescence band shifts [Eqns. (3.3) and (3.4)], and the values of µ g and µ e from Eqns. (3.7) and (3.8) can be given as [38] 1/ 2 3 m2 m 1 hca µ g = (3.9) 2 2m1 39

9 1/ 2 3 m1 + m 2 hca µ e = (3.10) 2 2m1 or m1 + m2 µ e = µ g ; (m 2 > m 1 ) (3.11) m m 2 1 The parameters m 1 and m 2 occurring for the difference (ν a -ν f ) and the sum (ν a +ν f ) of the wave numbers are linear functions of the solvent polarity parameters f (ε, n) and f (ε, n) +2g (n); and can be determined from the slopes of the straight lines. The method based on the empirical polarity scale proposed by Reichardt [28] gave towering results with solvatochromic shift of dipolar molecules that correlates much better with microscopic solvent polarity N E T rather than the traditionally used bulk solvent polarity functions involving dielectric constant (ε) and refractive index (n) as in the latter the error estimation of Onsager cavity radius a has been minimized. The theoretical basis for the correlation as spectral shift with N E T has been developed by Ravi et al. [16] and accordingly the excited state dipole moment is evaluated using the equation 2 3 µ ab N ν a ν f = ET + const. (3.12) µ B a where µ B =9 D and a B =6.2 Å are the dipole moment change on excitation and Onsager radius, respectively, for betaine dye and also are the corresponding quantities for the molecule of interest. N E T is defined using water and tetramethylsilane (TMS) as extreme reference solvents with the following equation [28] N ET ( solvent) ET ( TMS) ET ( solvent) 30.7 ET = = (3.13) E ( water) E ( TMS) 32.4 T T 40

10 The change in dipole moment is determined by m 81 µ = µ e µ g = (6.2 / a) 3 (3.14) where m is the slope of the linear plot of N E T vs. Stokes shift. III.2.2. Computational method Density functional theory (DFT) calculations were performed for the three probes Fluorescein 27(F27) and Sulfarhodamine B (SRB) to calculate ground and excited state dipole moments. The ground state geometries of theses molecules were fully optimized using DFT with popular hybrid B3LYP/6-31g* functional by Gaussian03 software [39]. However, information regarding the excited state dipole moments was obtained by optimizing the geometry in the excited state at CIS/6-31G* level using the ground state global minima as reference [40, 41]. III.3. III EXPERIMENTAL Chemicals used The laser dyes F27 and SRB were of the highest available purity procured from Lambda Physik GmbH, Germany and were used without further purification. All the solvents used in the study were procured from Fluka (HPLC grade) and were chosen as they are transparent and non-fluorescent in the range of excitation and fluorescence emission. III.3.2 Spectroscopic measurements Absorption spectra were recorded using UV-visible double beam ratio recording spectrophotometer (Hitachi, Model U-2800). Fluorescence spectrofluorometer (Hitachi, Model F-7000) was used to record the 41

11 fluorescence spectra. All the measurements were carried out at room temperature (298K) keeping the dye concentration very low (10-5 ~10-6 M) in order to avoid or minimize self-absorption and aggregation. The excitation wavelength used for F27 and SRB are 490 nm and 550 nm, respectively. The measurement of refractive index of probe solutions has been described in detail in Chapter II (section II.3). III.4. RESULTS AND DISCUSSION To examine the effect of the solute shape on the dipole moments in the excited state and the ground state, a number of solvents have been selected for the present study. Firstly, the non hydrogen-bond donating solvents (non-hbd solvents) such as acetone, acetonitrile, DMF and DMSO. Secondly, the hydrogen-bond donating solvents (HBD solvents) such as formamide, glycerol and alcohols are used which have been a subject of a number of studies related to polar solvation [42 and references therein]. Solvents covering wide range from non-hbd to HBD have been preferred. Thus, we have chosen various (i) aprotic (ε =21.01 to 47.24, from acetone to DMSO) and (ii) protic solvents (ε =7.93 to 33.6, from decanol to methanol, glycerol ε =42.5 and formamide ε =111). III.4.1. Evaluation of ground and excited state dipole moments F27 and SRB The molecular structures of F27 and SRB are shown in Fig.3.1. The values of absorption maxima λ max of F27 and SRB in the solvents used are given in Table 3.1. From this table, one can observe that the absorption maxima of the dyes are affected by the type of solvent and maximum shifts ( λ) for F27 42

12 and SRB are 23 and 18 nm, respectively for the solvents used in this work. Hence, this change in spectral position can be used as a probe for different types of interactions between the solute and solvents. The study of solvent effect based on spectral properties of dye solutions were carried out by using the spectral position in the solvents mentioned here and correlating these with the Kemlet-Taft solvent properties namely, π*, α, β, refractive index (n) and dielectric constant (ε), obtained from literature [43, 44]. As the shift in λ max values with solvent type reflects dye-molecule interactions, an attempt was made to study this fact exhaustively. The solvent parameters essential for this work are shown in Table 3.1. The spectral position of dye in a variety of solvents has revealed interesting results. Since all the solvents used in this study were polar in nature, one would expect that the solute would bind more strongly to a more polar solvent and consequently cause the spectra to shift to lower wavelengths. However, this is not observed from our results as λ max is lowest in the case of glycerol for F27 and acetone for SRB. This may be due to the reason that all other solvents used in this work are more polar than these two solvents in which λ max is lowest and can engage more strongly in a solventsolvent type of interaction or their ability to interact with the dye molecules reduces. These solvents, glycerol for F27 and acetone for SRB are less polar and interact with dye molecules in terms of dipole-dipole interactions, leading to a net stabilization of the ground state of the solute molecule and thus a hypsochromic shift in the spectrum of these solvents is observed. Alternatively, λ max value is shifted to lower energies in highly polar solvents such as 43

13 formamide because of strong solvent-solvent interaction or specific interaction between the solvent and hydrogen present in the functional groups of the dye molecule. Figs. 3.2(a) and 3.2(b) illustrate the plot of λ max versus the dielectric constant (ε) values in different non-hbd and HBD solvents for SRB dye. From this Fig., it can be seen that with increase in ε values, the spectrum is shifted to longer wavelengths. In case of F27, λ max of dye solution in DMSO (aprotic or non-hbd) and glycerol (protic or HBD) were found to be at lower wavelengths as compared to other solvents although their dielectric constants are higher among respective non-hbd and HBD solvents. This might be due to the formation of strong hydrogen bond between dye and solvent molecule. The variation of λ max versus π* in different non-hbd and HBD solvents for SRB shown in Fig. 3.3(a) and 3.3(b), wherein an increase in λ max values with π* specifies that dye interaction changes with increasing capability of a given solvent to form hydrogen bonds in solution. The effect of solvents on the absorption and emission spectra of SRB in aprotic (acetone to DMSO) and of F27 in protic (formamide and alcohols) solvents are shown in Fig.3.4 (a) and 3.4(b), respectively. The uncertainties in the measured wavelength of absorption and fluorescence maxima are ±0.5 nm and ±1 nm, respectively. The observed absorption and emission spectra of these two dyes are broad and they shift depending on the solvent used. The charge transfer band shows a shift of about 4-23 nm in the absorption spectra on changing the solvent from decanol to formamide for F27 and that in case of 44

14 SRB is 3-13 nm. A relatively larger spectral shift observed in the emission spectra as compared to the absorption spectra. A lesser variation in the absorption shift observed in all the solvents implies that the ground state energy distribution is not affected to a greater extent probably due to the less polar nature of these dyes in the ground state rather than the excited state. The pronounced shift in the emission clearly indicates that the dipole moment of the excited state is higher compared to that in the ground state. The large Stokes shift observed is also an indication of the charge transfer transition. The higher magnitude of Stokes shift suggests that the excited state geometry could be different from that of the ground state. A general observation is the increase in Stokes shift values with increase in solvent polarity indicating that there is increase in the dipole moment upon excitation. In such cases, the relaxed excited state S 1 will be energetically stabilized relative to the ground state S 0 and hence, a significant red shift of the fluorescence will be observed. The absorption data of dyes in different solvents was also analyzed in terms of various polarity scales. One among such methods involves the transformation of λ max of dyes in different solvents into molar transition energies [E T (dye), kcal/mole] by using the following equation [45], E T (dye) = 28591/ λ max (3.15) The E T (dye) values, obtained using equation (3.15), signify the transition energies that reflect the stabilization of the dye in its ground state in the respective solvent. This may perhaps be due to either hydrogen bond formation or dye - solvent interaction. Hence, E T (dye) gives a direct empirical 45

15 measure of dye solvation behavior. One can notice from Table 3.3 that, E T (dye) value is the highest as compared to other solvents in the case of glycerol for F27 and acetone for SRB. The reason for this being the same as described earlier. The wave numbers of absorption and fluorescence emission maxima of solutes along with the microscopic polarity scale E N T, are summarized in Table 3.3 for F27 and SRB. Consecutively, to estimate the ground state and excited state dipole moments of the dye molecules, the solvent polarity f(ε, n) and f(ε, n)+2g(n) parameters were calculated (Table 3.2). Figs.3.5(a) to 3.5(d) show the respective spectral shifts ν a -ν f and ν a +ν f for both the dyes which are observed in non-hbd solvents and HBD solvents against the polarity function f(ε, n) and f(ε, n)+2g(n), respectively. A linear regression was done and the data were fit to straight lines for both the plots whose slopes were taken as m 1 and m 2. For polar solutes like F27 and SRB, the interaction with non-polar solvents depends on the dipole-induced-dipole forces, while with aprotic (polar but non-hbd) solvents, the solute-solvent interaction depends on the stronger dipole-dipole forces. In HBD solvents (polar protic), in addition to dipole-dipole interaction, specific interaction such as H-bonding may be effective as the intermolecular charge transfer (ICT) character is favorable for H-bonding with hydroxyl groups present in protic solvents. Hydrogen bonding interaction usually puts a severe restriction on the validity of the eqns. (3.3) and (3.4). It is consequently useful as pointed out by others also [46, 47] to use E T (30) function which is the 46

16 empirical measure of the solvent polarity [48] for understanding the polarization dependence of spectral characteristics. The solvent polarity parameter E T (30), which also considers other interactions besides those of specific nature, was related with the absorption values. E T (30) values were acquired from literature for different solvents used in this study and are shown in Table 3.3. Fig. 3.6 depicts the correlation between the absorption value (in wavenumber) and E T (30) for F27 dye. Linear correlation of absorption energy over a range of E T (30) represents the presence of specific interactions between the solute and solvents. Unfortunately, E T (30) values have the dimension of kcal/mol, a unit which should be discarded in the framework of SI units [28]. For that reason, the use of the so-called normalized N E T values have been suggested, as defined in eqn. (3.13). Figs. 3.7(a) and 3.7(b) show the plots of Stokes shift as a function of N E T in all the solvents for F27 and SRB, respectively. The linear N E T dependence of Stokes shift reveal the existence of general type of solute-solvent interaction in which the Stokes shift depends on dielectric constant and refractive index of the solvents. The Onsager s cavity radius is calculated using both Edward s increment method [49] and from Suppan equation [50]. These two methods collectively yield almost the same cavity radius of 4.15Å and 4.93Å for F27 and SRB, respectively. Using eqns. (3.9) and (3.10) the ground and excited state dipole moments are evaluated and summarized in Table 3.4 along with the slopes m 1 and m 2. The difference in dipole moment calculated from solvent perturbation method and the one calculated using eqn. (3.14) are reasonably in 47

17 good agreement in case of protic solvents, obviously indicating the excited state dipole moment to be higher compared to ground state. The observed ground state dipole moment of F27 and SRB are 2.65D and 6.85D in aprotic solvents 2.20D and 5.61D in protic solvents, respectively. The disparity in the values of dipole moments of F27 and SRB can also be explained on the basis of their possible resonance structures as shown in Figs.3.8 (a) and 3.8 (b). In case of F27 dye, non-bonding electrons on the oxygen of pyran ring, hydroxyl group, carbonyl group and carboxylic acid group along with chlorine contribute towards the mobility of electrons on the aromatic ring. Alcohols form strong hydrogen bonding, thus oxygen atom of OH can better contribute to the resonance structure. On excitation, the oxygen atom of the carbonyl group and that of pyran ring also contribute towards the mobility of electrons on the aromatic ring group by delocalizing their nonbonding electrons. Whereas SRB (anion), is a model hydrophilic probe and an ionic compound. The π electron mobility is more in SRB because of more electron tendency of nitrogen atom. The nitrogen atom of diethylamine group in SRB has lone pair of electrons. These non-bonding electrons on the nitrogen atom of tertiary amino group N (C 2 H 5 ) 2 contribute towards the mobility of electrons on the aromatic ring. Since the nitrogen atom of tertiary amino group is sp 3 hybridized, its electron donating tendency is more. Upon excitation the tertiary amino group becomes strong electron donor. This is the reason why SRB possesses higher dipole moment than F27. 48

18 The ground and excited state dipole moments of F27 and SRB probes were also determined from ab initio calculations using DFT [51] and CIS method, respectively (Table 3.4). The ground state optimized geometries of these probes acquired using B3LYP functional with 6-31g * basis are shown in Fig.3.9 (a) and 3.9 (b). The arrow in the Fig. indicates the direction of transition dipole moment in the ground state. It is evident from Table 3.4 that the ground state dipole moments evaluated from Salvatochromic shift method are smaller than those from ab initio calculations for F27 and SRB probes. Numerous reports in literature have shown disagreement between theoretical and experimental values [52]. The excited state dipole moments were computed using CIS method to estimate the minimum of the lowest excited singlet state and optimized using 6-31g *. The ground and excited state dipole moments obtained from ab initio methods using DFT is shown in Table 3.4 for the comparison with the experimental results. The ground state dipole moments, calculated using B3LYP/6-31 g* basis set for F27 and SRB respectively, are 6.19 D and D. The ground state optimized geometry of F27 and SRB along with total atomic charge distribution is shown in Fig.3.9. In the calculation of excited dipole moment, CIS/6-31 g* is used and are found to be 8.26 D and D in case of F27 and SRB, respectively. However, higher values in the excited state dipole moments were noticed in case of theoretical values compared with experimental one. These results were unanticipated. Several authors have observed disagreement between theoretical and experimental values [52, 53, 54]. Aaron and Maafi [52] reported disagreement between experimental and theoretical values in the excited state for acridine, acridine yellow and 9-49

19 aminoacridine. These differences found by Aaron and Maafi [52] and also by Sharma et al. [8] could be partly due to the simplification used in Bakshiev and Kawski-Chamma-Viallet s method and to strong specific effects of the solvents of different nature. 50

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24 Table 3.4. Dipole moments, slopes (m 1 and m 2 ) and correlation factor (r) of F27 and SRB molecules: Molecules Radius (Å) Solvents µ g (D) µ e (D) µ (D) µ e /µ g m 1 (cm -1 ) m 2 (cm -1 ) r F Aprotic (1.010 * ) :0.864 Protic (2.596 * ) : a 8.26 b SRB 4.93 Aprotic (1.101 * ) :0.978 Protic (2.115 * ) :0.881 * Calculated using equation 14. a Calculated from B3LYP functional with 6-31g * basis. b Excited state dipole moment using CI singles (CIS) method a b

25 Table 3.3 Some physical constants of solvents and spectral data of F27 and SRB # Solvents ( ) T E dye 1 (30) E T #1 N E # T ν a (cm -1 ) ν f (cm -1 ) F27 SRB F27 SRB F27 SRB Acetone Acetonitrile DMF DMSO Formamide Glycerol Methanol Ethanol Propanol Butanol Pentanol Hexanol Heptanol Octanol Nonanol Decanol E T (30) values for both general and alcohols solvents are from Ref. [1,45, 46]. 1 Solvent polarity in the unit of kcal/mol. N E T values for both general and alcohols solvents are from Ref. [1].

26 Table 3.2. Calculated values for solvent polarity parameters f (ε, n), g(n) and f (ε, n)+2g(n) Solvents f (ε, n) 2g (n) f(ε, n)+2g(n) Acetone Acetonitrile DMF DMSO Formamide Glycerol Methanol Ethanol Propanol Butanol Pentanol Hexanol Heptanol Octanol Nonanol Decanol

27 Table 3.1 Some physical constants of solvents and absorption maxima of F27 and SRB in various solvents Solvents n # ε # π *# α # β # λ max (nm) F27 SRB Acetone Acetonitrile DMF DMSO Formamide Glycerol Methanol Ethanol Propanol Butanol Pentanol Hexanol Heptanol Octanol Nonanol Decanol # The values of π*, ε, α and β for general solvents and alcohols are from taken from Ref. [45, 46]. The values of n are taken from Fluka Catalogue for general solvents and alcohols.

28 Figure 3.9 (a) Ground state optimized structures of F27 molecules obtained using B3LYP functional with 6-31g * basis is shown along with the Distribution of charges. The arrow indicates the direction of dipole moment.

29 Figure 3.9 (b) Ground state optimized structures of SRB molecules obtained using B3LYP functional with 6-31g * basis is shown along with the distribution of charges. The arrow indicates the direction of dipole moment.

30 HO O O HO O O Cl OH Cl Cl OH Cl O O HO O O HO O O Cl Cl Cl Cl OH OH O O HO O O HO O O Cl Cl Cl Cl OH OH O O (a) Figure 3.8 Possible resonance structures of (a) F27.

31 (C 2 H 5 ) 2 N O N(C 2 H 5 ) 2 SO 3 SO 3 Na (C 2 H 5 ) 2 N O N(C 2 H 5 ) 2 SO 3 SO 3 Na (b) Figure 3.8 Possible resonance structures of (b) SRB.

32 1200 (a)f ν a -ν f (cm -1 ) NHBD ( ) HBD ( ) E T N 1200 (b)srb 900 ν a -ν f (cm -1 ) NHBD (R= ) HBD (R= ) E T N N Figure 3.7 Plot ν a -ν f vs E T of (a) F27 and (b) SRB in all solvents studied.

33 19800 F Wavenumber (cm -1 ) R= E T (30) Figure 3.6 Plot of absorption value of F27 (in wave number) vs. E T (30) in all solvents studied.

34 2250 f(ε,n)+2g(n) (a) F27NHBD ν a -ν f (cm -1 ) ν a +ν f (cm -1 ) f(ε,n) f(ε,n)+2g(n) (b) F27 HBD ν a -ν f (cm -1 ) ν a +ν f (cm -1 ) f(ε,n) Figure 3.5 Plot of ν a -ν f vs f(ε, n) and ν a +ν f vs f(ε, n)+2g(n) of F27 in (a) non-hbd (b) HBD solvents.

35 (c) SRB NHBD f(ε,n)+2g(n) ν a -ν f (cm -1 ) ν a +ν f (cm -1 ) f(ε,n) f(ε,n)+2g(n) (d) SRB HBD ν a -ν f (cm -1 ) ν a +ν f (cm -1 ) f(ε,n) Figure 3.5 Plot of ν a -ν f vs f(ε, n) and ν a +ν f vs f(ε, n)+2g(n) of SRB in (c) non- HBD and (d) HBD solvents studied.

36 1.0 (a) SRB NHBD Absorption Acetone 2.DMF 3.Acetonitrile 4.DMSO λ(nm) (b)f27 HBD Absorption Formamide 2.Methanol 3.Ethanol 4.Propanol 5.Octanol Fluorescence(a.u.) λ (nm) Figure 3.4 Absorption and fluorescence spectra of (a) SRB in non-hbd and (b) F27 in HBD solvents.

37 (a)srb NHBD π Acetone 2.Acetonitrile 3.DMF 4.DMSO λ (nm) 0.96 (b)srb HBD π λ (nm) Figure 3.3. (a) Absorption shift of SRB dye solution as a function of solvent polarizibility(π*) in non-hbd solvents. (b) Absorption shift of SRB dye solution as a function of solvent polarizibility(π*) in HBD solvents.

38 100 (a) Dielectric constant Wavelength(nm) (b) Dielectric constant Wavelength(nm) Figure 3.2: (a) Absorption shift of SRB dye solution as a function of dielectric constant in non-hydrogen-bond donating solvents. (b) Absorption shift of SRB dye solution as a function of dielectric constant in hydrogen-bond donating solvents.

39 HO O O Cl Cl COOH (a) (H 5 C 2 ) 2 N O N + (C 2 H 5 ) 2 SO 3 - SO 3 Na (b) Figure 3.1: Molecular structures of (a) Fluorescein 27 (b) Sulforhodamine B

40 100 (a)srb NHBD 80 Dielectric constant Acetone 1 2.Acetonitrile 3.DMF 4.DMSO Wavelength(nm) (b) SRB HBD 90 Dielectric constant Wavelength(nm) Figure 3.2. (a) Absorption shift of SRB dye solution as a function of dielectric constant in non-hydrogen-bond donating solvents. (b) Absorption shift of SRB dye solution as a function of dielectric constant in hydrogen-bond donating solvents.