Demonstration of Solute-specific Synergism in Binary Solvents

The structure and solvation behavior of binary liquid mixtures of Methanol (MeOH) and N, N-Dimethylformamide (DMF) are explored by ascertaining their intermolecular interactions with either Rhodamine-B (RhB) or Rhodamine101 (Rh101) dye through steady-state absorption, emission, and two-photon induced fluorescence. Specifically, in the present investigation, we examine the strong synergistic solvation observed for the combinations of hydrogen bond donating (MeOH) and accepting (DMF) solvent pairs. Solvatochromism causes the solvatochromic probe molecules to sense increased polarity compared to their bulk counterparts. The origin of synergism was explained in terms of solute–solvent and solvent–solvent interactions in binary solvent mixtures interactions, as evidenced by probe dependence. The solvation behavior of the Methanol and DMF binary solvent mixture shows strong probe dependence, with Rh101 showing synergism while RhB does not.


Introduction
Rates of chemical reactions and their equilibrium positions, as well as the position and intensity of absorption bands in UV/ Vis/near-IR, IR, ESR, and NMR spectroscopy, are solventdependent [1][2][3][4]. The chemical physics of solvation in solvent mixtures is of current interest [5][6][7]. Experimental evidence suggests that the solute may change the solvent composition in the solvation sphere compared to that in bulk [8,9]. This phenomenon is termed 'preferential solvation' and originates due to a difference in solvent-solvent and solute-solvent interaction. Apart from preferential solvation, there is always some probability that both constituent solvents can solvate the molecules together by mutual interaction and give rise to a completely different environment. Such a phenomenon is observed for some specific solvent pairs and is termed 'Synergism'. Synergism has been studied for binary solvent mixtures experimentally [7,[10][11][12][13], and theoretically [14][15][16][17] regarding solvent-solvent and solute-solvent interactions. An example of this phenomenon is the synergistic solvation, which can result in a higher/lower reaction rate [18][19][20], higher solubility of solutes [20], or higher product yield [21] in the solvent mixture than in each of the pure solvents. Synergistic solvation can be detected by measuring the absorption spectrum of the solvatochromic probe molecule, which in this case senses increased polarity of the mixture (reflected by longer absorption wavelength), compared to the pure components, as observed in the mixtures of MeOH with hydrogen-bond donating solvents DMF [22][23][24]. In that case, a solvent exchange model was applied to the mole fraction dependence of the transition energy of the solvatochromic indicator to show that the synergistic solvation prevails over the preferential solvation. Electronic spectroscopy provides a suitable method for studying solvation. It has been observed that the maximum energy (E T ) of electronic transition involving intramolecular charge transfer in various solutes depends significantly on the local environment around the solute [1,10]. At the microscopic level, information about solvent-solvent and solute-solvent interactions consists of nonspecific and specific solvation modes. Usually, three parameters, solvent polarity-polarizability, H-bond acceptance ability or solvent basicity, and H-bond donation ability or solvent acidity, have been characterized to represent the various modes of solvation [1,4,8]. Mancini et al. [18] proposed that the combination of bipolarity and hydrogen bond donating and accepting capacity of solvents leads synergistic effect in binary solvent mixtures, which is reflected through E T (30) empirical solvent polarity parameter.
Characterization of solvation behavior has been done by employing various methods. The different modes of solvation of solvatochromic molecules have been widely investigated by various ultrafast techniques such as picosecond or femtosecond time-resolved fluorescence spectroscopy [25][26][27][28][29][30][31], two-dimensional IR spectroscopy [32], femtosecond pump − probe spectroscopy [32][33][34][35], dielectric relaxation spectroscopy [25]. Bhattacharyya et al. [36] measured the thermal-lens signals as a function of the relative proportion of binary solvent mixtures. Additionally, much work has also been carried out using molecular dynamics simulations [26,32], vibrational spectroscopy [37], UV-Vis spectroscopy [7,11], NMR chemical shifts [19,38], solvation dynamics [27], etc. Refractive index measurements of binary solvent mixtures have also been used to determine the quantitative information about the solvent-solvent interactions [39,40]. Other related kinds of solvatochromic species have been used as probes(solute) of their environment in micelles [41][42][43], polymers [44,45], surfaces [44,46], zeolites [47], and inorganic glasses [48,49], etc. They may be used in this way either by their response to the electric field experienced in a particular environment or because of specific interactions between the probe (solute) and the environment(solvent), e.g., hydrogen bonding between solvent-solvent and solute-solvent. Solvatochromism is also a valuable predictor of nonlinear optical behavior since the properties that give rise to high solvatochromism are the same for significant second-order nonlinear optical coefficients (β) [45,50].
Despite a large number of studies, the experimental determination of the nature of the solvation cage in the binary solvent mixture has not yet been well established. In the present contribution, we throw light on the origin of synergism in the binary solvent mixture of MeOH and DMF in RhB and Rh101 using electronic spectroscopy, fluorescence spectroscopy, and Two-Photon Induced Fluorescence (TPIF) [44].
In the two-photon absorption process, a molecule or fluorophore absorbs two photons simultaneously, and no change occurs in the angular momentum of the material. The two-photon absorption process is a third-order nonlinear process [51]. The two-photon laser scanning microscopy (TPLSM) was first carried out by Denk et al. [52] at Cornell University, Ithaca, New York, which has led to an increasing interest in the field of microscopy. TPLSM has shown vast applications in the field of medicinal and biological research. Kaiser and Garret, in 1961, first demonstrated the two-photon induced fluorescence in the CaF 2 :Eu [2] + crystal [53]. Two-photon induced fluorescence (TPIF), an indirect and accurate method for calculating the two-photon absorption cross-section provided quantum efficiency of the fluorophore, is already known to all. One of the advantages of using the TPIF method is that we can avoid the influences of other optical nonlinear processes in determining the two-photon absorption cross-section (TPACS) of materials so that obtained values will be very close to the accurate ones. Wide applications of materials with high TPACS have been reported through investigation, such as three-dimensional micro-and nanofabrication [54,55] three-dimensional fluorescence microscopy [56], optical limiting [57,58], and optical data storage [59]. However, the surrounding environment of solvent molecules can influence this twophoton process in a fluorophore.
The TPIF process is mainly due to the polarity of the medium, which significantly modulates the TPACS values [60,61]. Many articles have been published supporting the effects of the surroundings of single solvent molecules on TPACS values. Our research group has already published the effects on the values of two-photon absorption and twophoton fluorescence using single solvent molecules of several Xanthenes dyes [62]. A recent study has noted that the field of binary solvents for understanding intermolecular interactions has gained interest from many researchers. The importance of binary solvent mixtures in chemical, physical, and biochemical processes has been extensively discussed in previous literature [2,32,62]. Thus, keeping the recent approaches in mind, our research group had already published an article on the effect of different sets of Binary Solvents of Rhodamine-6G Dye on Two-Photon Induced Fluorescence [63]. We tried to compare the effects of intermolecular interactions on the TPIF of commonly used fluorescent dyes RhB, which is often used as a tracer dye within water to determine the direction and rate of flow and transport, and Rh101, which is a highly stable fluorophore in the Rhodamine family. A microheterogeneous environment can be created for dye molecules by mixing two miscible solvents. Research in binary solvents draws considerable interest because of its widespread applications in areas such as low-temperature glasses [64], cryo-protecting agents [65], and observing the antagonistic effect on a protein structure [64]. We have used different sets of binary mixtures in various fractions of Methanol as a standard component with each of the solvents, water, DMF, and chloroform to compare the information about the effects of intermolecular interactions on the TPIF of RhB and Rh101.

Material
We used Fluorescence grade Rhodamine 6G dye (dye content:99%) (used as the sample reference), Rhodamine B dye (dye content:99%), and Rhodamine 101 dye (Dye content:99%) that were purchased from Sigma-Aldrich and used without further purification. HPLC-grade Methanol (MeOH) (purity ≥ 99.7%) was purchased from Thomas Baker (Mumbai, India), HPLC UV-grade. N, N-Dimethylformamide (DMF)(purity ≥ 99.9%) was purchased from SRL (Mumbai, India). All care was taken to avoid moisture contamination during the experiments. All the experiments were performed at room temperature.

Steady-state Absorption and Fluorescence Measurements
All the one-photon UV-VIS absorption spectra of RhB and Rh101 in the binary solvents, as well as in the individual pure solvents in 10 -4 M concentration, are measured in 1 mm long quartz cuvette through the Perkin Elmer Lambda 900 UV/VIS/NIR spectrometer (a double-beam, double monochromator ratio recording system with pre-aligned tungstenhalogen and deuterium lamps as sources). Single-photon fluorescence spectra were recorded using Perkin Elmer LS55 Fluorescence Spectrometer. Quartz Fluorescence cuvettes (10 × 10 mm) were used with a white PTFE stopper to prevent solvent evaporation. Emission spectra were background corrected using a solvent blank to correct any emission arising from the solvent.

Two-Photon Induced Fluorescence (TPIF)
In this experiment, we used the Mode-locked amplified Femtosecond Ti:Sapphire laser (Astrella, Coherent Inc. USA) to study two-photon induced fluorescence (TPIF). Figure 1 illustrates a representative schematic of the TPIF setup. We measured the pulse width of the laser beam using the field autocorrelation technique to be 49 fs. We used a 1 kHz repetition rate pulsed laser beam for the excitation of the sample at 802 nm. This laser beam was focused into Quartz Fluorescence cuvettes (10 × 10 mm) using a 20 cm focusing lens. The sample cuvette was placed perpendicularly to the propagation direction of the laser beam so that the maximum TPIF was collected through a collecting objective (10 × 0.25 NA, Newport). The TPIF thus collected was focused onto an optical fiber connected to the HR2000 spectrometer. The HR2000, in turn, is connected to the computer through the GPIB interface. Data was collected using the standard LabVIEW program with 1000 ms of integration time of the HR2000 spectrometer. We carried out the power-dependent TPIF experiments to verify the two-photon absorption process. For such power-dependent studies, we used the combination of a polarizer and half-wave plate for the smooth variation of intensity of the laser where the change of the laser beam intensity was controlled by the half-wave plate while keeping the polarizer undisturbed.

Single Photon Absorption and Fluorescence Spectra of RhB and Rh101 Dyes in the Binary Solvent Mixtures of MeOH and DMF
RhB([9-(2-carboxyphenyl)-6-(diethylamino)xanthen-3-ylidene]-diethylazanium chloride) is a cationic and Rh101(2-(3-oxa-23-aza-9-azoniaheptacyclo [17.7.1.15,9.02,17.04,15.023,27.013, 28] octacosa-1 (27),2(17), 4,9(28), 13,15,18-heptaen-16-yl)benzoate) is inner salt dyes, whose chemical structure are shown in Fig. 2a, b. It has a large number of conjugated double bonds that enhance its two-photon absorption process. Absorption study of RhB and Rh101 dye in binary mixture solvents and pure individual component solvents exhibit a doublet band with one absorption maxima and a shoulder in the main peak. Positions of these doublet bands are directly influenced by the nature of the surrounding solvent molecules. The occurrence of the shoulder in the main absorption peak is directly related to the formation of aggregates of dye molecules we can see in, Fig. 3a, b,   Fig. 1 Schematic diagram of TPIF setup which in turn, is directly related to the nature of the solvents viz. polar or nonpolar. We took the absorption spectra for RhB and Rh101 in binary mixtures of MeOH and DMF (MeOH + DMF) by varying volume fractions. We can see from Fig. 3a, b that the shoulder in the main peak of RhB in MeOH + DMF decreases as the mole fraction of MeOH increases, whereas in the case of Rh101 dye, the main peak of Rh101 in MeOH + DMF increases with the increasing mole fraction of MeOH.
The alteration of the absorption maximum and emission max of either dye (RhB or Rh101) in the MeOH + DMF binary set of solvent as a function of X MeOH is presented in Fig. 4a, b, respectively. To represent the stabilization of the dye in the corresponding binary mixture concept of molar electronic transition energy (E T ) has been introduced, whose relationship to the absorption maximum (λ max , nm) is given by [1,19,[65][66][67][68][69][70][71][72][73].
For an ideal mixture, one expects that any equilibrium property in a binary liquid (e.g., solvation energy, (1) E T = 28591 max composition) will be a weighted average of the values in the two solvents. Thus, in an ideal case, where all solvent − solvent interactions are assumed to be the same, the dye is characterized by a maximum value of the molar electronic transition energy given by [6,27,29].
where, X S 1 and X S 2 , represent mole fractions of solvents 1 and 2, respectively and E TS 1 and E TS 2 are the E T values of the dye or indicator of solute in pure solvents 1 and 2, respectively. This equation corresponds to the ideal behavior of a binary solvent mixture, and the variation of E T as a function of the mole fraction of either component should always be linear. However, in practice, the variation is barely linear, suggesting specific and selective interactions between the dye molecule and one of the components of the solvent mixture; this is termed 'preferential solvation'. In addition, deviations from the ideal behavior might also appear from the interaction of the combined solvent counterparts in the binary solvent mixture, exhibiting a synergistic effect. A direct measure of stabilization energy is given in terms of   Fig. 4 that RhB in MeOH + DMF shows a regular pattern of variation in absorption maximum and shows a blueshift. In contrast, the absorption maximum of Rh101 in MeOH + DMF binary solvents shows redshift and then blueshift after X MeOH = 0.6.
Thus, it is clear from Fig. 4 that the emission maximum of dyes in a different set of solvents strongly depends on the composition of the solvent mixture. Emission maxima of each dye, RhB, and Rh101, show systematic opposite solvation behavior with consistent emission maxima pattern shifts observed in MeOH + DMF, i.e., from 585 to 580 nm for RhB and from 592 nm to 599.5 nm in the case of Rh101.

Two-photon Induced Fluorescence of RhB & Rh101 Dyes in Different Sets of Binary Solvents Mixture
We carried out two-photon induced fluorescence of RhB and Rh101 in MeOH + DMF binary solvents at varying volume fractions of Methanol (X MeOH ). Also, we compared it with respective single-photon fluorescence spectra. Figure 5a, b shows the two-photon induced fluorescence spectra for the RhB and Rh101 in the pure Methanol at various excitation laser powers at 802 nm. Figure 5c, d shows the Log-Log plot of the TPIF intensity versus excitation laser powers, which verifies that the fluorescence emission originates from the two-photon absorption process related to the linear absorption spectra. The interactions between the molecules of the dye and solvent directly affect the energy levels between the ground and excited states. Fluorescence emission always occurs from the first excited singlet state, leading to the spectral shift in the spectra towards longer wavelengths, i.e., redshift. Generally, the Lippert-Mataga equation is used to describe the solvent dependent spectral-shift, as given below [74].
where ν A and ν F are the absorption and emission maximum in wave-number units h = Planck's constant, c = velocity of light, and a = radius of the cavity in which the sample fluorophore is kept. ε and n are the solvent's dielectric constant and refractive index, respectively. As can be seen from the two-photon induced fluorescence (TPIF) spectra, peak wavelengths are shifted from their corresponding linear absorption spectra in pure solvents and MeOH-DMF binary solutions. From the two-photon induced fluorescence signal, we can determine the two-photon excitation action cross-section, σ*, which is directly proportional to the two-photon absorption cross-section, σ 2, according to the following equation where ∅ is the fluorescence quantum yield. Two-photon excitation action cross-section (TPEACS, σ*) values were estimated using the following relation [75].
where "ref" stands for the reference (here, R6G in water was used as the reference) and "s" stands for the sample. C i is the concentration in moles/liter, n i is the refractive index, and as the σ ref [43]. All the analyses were performed using Mathematica 9. Fluorescence quantum yield ∅ was calculated by using the following relation [76]: where A i is the absorbance, and D i is the integrated area under the corrected (baseline subtracted) emission spectrum.
We have used the reported fluorescence quantum yield of R6G ( ∅ Rh6G ~ 0.95) in water as the standard reference [77,78]. We used the Lorentz-Lorenz Eq. [36,79] to calculate the refractive indices of binary solvents.
where n 1 and n 2 are the refractive indices of pure components, n is the refractive index of the binary solvents, and x 1 , x 2 , and ρ 1 , ρ 2 are mole fractions and densities of components 1 and 2, respectively. After rearranging and applying some manipulation to the above equation, the refractive index of the binary solvents can be determined by the following equation: where L is given by: Also, the density of binary solvent can be given by: where, V X 2 represents the volume fraction of the solute in the binary solvent mixture.
All the solutions of RhB and Rh101 were prepared at 10 -4 M concentration in MeOH + DMF binary solvents and the corresponding individual component solvents. As we see from Fig. 6a, in MeOH + DMF mixtures, with increasing mole fraction of MeOH (X MeOH ), RhB shows a redshift X MeOH = 0.00 to 0.1 (TPIF λ max increases from 569.91 nm to 596.81 nm). After that, there is a consistent blueshift (TPIF λ max decrease from 596.81 nm to 589.65 nm), i.e., there is a formation of non-fluorescent aggregation (dimmers, trimmers, etc.), which reaches a maximum at X MeOH = 0.1. In the case of Rh101, the initial redshift is seen at mole-fraction X MeOH = 0.00 to 0.4 (TPIF λ max increases from 575.75 nm to 612.02 nm). After that, there is a blueshift X MeOH = 0.40 to 1.00 (TPIF λ max decreases from 612.02 nm to 607.10 nm), i.e., the formation of non-fluorescent aggregation maximum for Rh101 occurs at X MeOH = 0.30-0.40. Reduction in aggregation formation in RhB and Rh101 dyes directly influences the TPIF intensity and results in higher TPEACS values Fig. 7 towards higher volume fractions of Methanol.
Further, as we can see from Fig. 7, at binary mixtures, in MeOH + DMF, RhB shows the highest TPEACS value at X MeOH = 0.80 (24.16 GM) and lowest at X MeOH = 0.40 (5.71 GM) whereas Rh101 is showing the highest TPEACS value at X MeOH = 0.60 (21.73 GM) and lowest at X MeOH = 0 (1.74 GM). The reason for showing this enhancement in TPEACS value in the MeOH + DMF binary mixture may be correlated with the non-selective solvation in MeOH + DMF called synergism [18]. This behavior occurs because of networking between weak H-bond and H-bond-donator MeOH and H-bond acceptor DMF, which gives rise to different solvent responses. An earlier study of the MeOH + DMF binary mixture reported [80] that at the 1:1 ratio of the components, there is the formation of a stable "complex" (sub-unit) of the type MeOH + DMF. The complex structure formation might be the reason for showing lesser TPEACS. Based on the above observations and arguments, we can propose that weaker intermolecular interactions between components of the binary solvent mixtures are giving rise to higher two-photon processes and, consequently, higher TPEACS values of the binary mixture solvents. Thus, Rh101 explores the synergism in the MeOH + DMF binary mixtures while RhB does not and instead shows preference towards solvation.

Conclusions
Steady-state absorption, emission, and two-photon induced Fluorescence Spectra of RhB and Rh101 dyes in MeOH + DMF binary solvent mixture explore the structure and solvation behavior of binary solvent mixtures. Strong synergistic solvation was observed for the combinations of hydrogen bond donating (e.g., MeOH) and accepting solvent (e.g., DMF) pairs, which causes solvatochromic probe molecules to sense increased polarity compared to the bulk counterparts. The origin of the strong synergism was explained in terms of solvent-solvent interactions as evidenced by probe dependence single photon absorption, emission, and two-photon induced Fluorescence measurements. The solvation behavior of the MeOH + DMF binary mixture shows strong probe dependence with no synergism observed when RhB was taken as the solvatochromic probe, ascribed to the higher ground state dipole moment of RhB (17.3642 D) relative to Rh101 (7.0186 D) ( Table 1 in S.I.). Such strongly perturbed systems (due to high dipole moment) do not allow the persistence of the hydrogen bonding network, resulting in preferential solvation instead of synergism.