Solvatochromism and ZINDO-IEFPCM solvation study on NHS ester activated AF514 and AF532 dyes: Evaluation of the dipole moments European Journal of Chemistry 13 (1) (2022) 8-19 European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2022 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. https://dx.doi.org/10.5155/eurjchem.13.1.8-19.2123 European Journal of Chemistry View Journal Online View Article Online Solvatochromism and ZINDO-IEFPCM solvation study on NHS ester activated AF514 and AF532 dyes: Evaluation of the dipole moments Mallikarjun Kalagouda Patil 1, Mare Goudar Kotresh 2, Tarimakki Shankar Tilakraj 1 and Sanjeev Ramchandra Inamdar 1,* 1 Laser Spectroscopy Programme, Department of Physics, Karnatak University, Dharwad 580003, India 2 Department of Physics, Vijayanagara Sri Krishnadevaraya University, Bellary 583104, India * Corresponding author at: Laser Spectroscopy Programme, Department of Physics, Karnatak University, Dharwad 580003, India. e-mail: him_lax3@yahoo.com (S.R. Inamdar). 10.5155/eurjchem.13.1.8-19.2123 Received: 07 May 2021 Received in revised form: 23 October 2021 Accepted: 01 November 2021 Published online: 31 March 2022 Printed: 31 March 2022 In this study, the solvatochromic effect on the photophysical properties of Alexa Fluor 514 (AF514) and Alexa Fluor 532 (AF532) fluorescent dyes is examined experimentally and computationally. To explore the solvatochromism and dipole moments, the steady-state absorption and fluorescence spectra of the dyes were measured in a series of organic solvents. Various solvent correlation models, like Bilot-Kawski, Lippert-Mataga, Bakhshiev, Kawski-Chamma-Viallet, and Reichardt microscopic solvent polarity parameters, were adapted to determine the dipole moments in their ground and excited states. For the computational investigation, the ground and excited-state geometries are optimized using density functional theory (DFT) and time-dependent density functional theory (TD-DFT), respectively, in vacuum. Furthermore, semiempirical ZINDO with the IEF-PCM model is used to evaluate the absorption transition energies of these dyes, which are comparatively studied in various solvent polarity along with experimental data. Additionally, the highest occupied molecular orbital energies (HOMO) and lowest unoccupied molecular orbital energies (LUMO), chemical softness, chemical hardness, energy gap, chemical potential, electronegativity, and molecular electrostatic potential (MEP) were estimated using DFT calculations at the CAM-B3LYP/6-311G(d,p) level, in gas phase. The experimental and computational results reveal that the singlet excited state dipole moment is greater than that of the ground state for the molecules considered. The angle between ground- and singlet excited-state dipole moments are found to be 0.50 and 0.49° making them almost parallel to each other. The natural bond orbital analysis (NBO) has been employed to investigate the stability of the molecule, inter- and intra-hyper-conjugative interactions and charge delocalization within the molecule. DFT TD-DFT Dipole moment Alexa Fluor dyes Solvatochromism Natural bond orbital Cite this: Eur. J. Chem. 2022, 13(1), 8-19 Journal website: www.eurjchem.com 1. Introduction In the last few years, Alexa Fluor dyes and their bio- conjugates have generated remarkable interest in the field of surface energy transfer (SET), fluorescence resonance energy transfer (FRET), fluorescence bioimaging, and biosensing [1-4] due to their strong absorption spectra spanning the entire visible region, bright and photostable fluorescence, high quantum yields, long fluorescence lifetime, good water solubility and insensitivity of their absorption and fluorescence spectra over a broad range of pH values. Alexa Fluor 514 (AF514) and Alexa Fluor 532 (AF532) (Figure 1), respectively, are green and yellow fluorescent dyes that have been used successfully for analyzing/detecting analytes [5], detecting chromosomal aberrations [6,7], imaging cells [8,9], labeling amines/amino groups [10], labeling/detecting nucleic acids [11,12], probing the charge-transfer dynamics in DNA [11], and quantifying proteins etc. [13] The spectral behavior of the solute molecule strongly depends on physical intermolecular solute-solvent interactions (such as ion-dipole, dipole-induced dipole, and hydrogen bonding, etc.). The intermolecular force between the solute solvent depends on the physical properties of the solvent molecule, such as polarity, polarizability, dielectric constant, etc. There are important environmental factors that influence the position, intensities, and shape of the electronic spectra of the fluorophore. The ground and excited-state dipole moments of the fluorophore disclose information on its electronic and geometrical structure and the sharing of electrons in the relevant states. Therefore, studying the effect of solvents on the spectral behavior and dipole moments helps in extracting information about the excited state activities of the molecule. For the estimation of singlet excited-state dipole moment, various techniques are available such as electronic polarization of fluorescence [14], electronic dichroism [15], microwave conductivity [16], and stark splitting [17,18], but their use is restricted to comparatively simple molecules. Solvatochro- mism is one of the most widely used approaches to understand the effect of solvent and charge distributions in both ground and excited states [19-21]. ABSTRACT RESEARCH ARTICLE KEYWORDS https://dx.doi.org/10.5155/eurjchem.13.1.8-19.2123 https://www.eurjchem.com/ https://dx.doi.org/10.5155/eurjchem.13.1.8-19.2123 mailto:him_lax3@yahoo.com http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.13.1.8-19.2123&domain=pdf&date_stamp=2022-03-31 Patil et al. / European Journal of Chemistry 13 (1) (2022) 8-19 9 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.8-19.2123 O C O ON O O SO3SO3H H N NH2 C O OH CH3 H3C CH3 (a) O H N H N C O ON O O CH3 CH3 SO3SO3H H3C H3C CH3 CH3 (b) Figure 1. Molecular structures of Alexa Fluor 514 (a) and Alexa Fluor 532 dyes (b). The dipole moment of fluorophore gives insight into the distribution of charge around the probe, the electron density, and the structure of the dye in solution. Hence, solvatochro- mism is an experimental endorsement of variations in the spectral behavior during electronic transitions and acts as important evidence for intramolecular charge transfer (ICT) in an excited state, and it plays an extensive role in research. In our previous studies, we have reported the computational and experimental investigations of solvent effect on Alexa Fluor 350 dye in a variety of solvent environments [22]. The effect of solvent polarity on the spectral behavior of the solute results in the shifting of the spectra depending on the fluorophore and the nature of the transition (σ → σ*, n → σ*, π → π*, and n → π*). The solvatochromic shift method is helpful to assign electronic transitions, in particular, π → π* or n → π*. The transitions corresponding to π → π* or n → σ* are named positive and negative solvatochromism corresponding to bathochromic (red) and hypsochromic (blue) shift with increasing polarity of the solvents, respectively. The mixing of π → π* or n → π* transitions, leads to variations in the magnitude of dipole moments [23,24]. To explore various solvent properties, dielectric constant, refractive index, induced dipole moment, and relative permittivity, we have employed numerous methods through qualitative solvatochromism. Dimroth and Reichardt reported a simple approach to discriminate solvatochromism effectively in terms of an empirical solvent polarity parameter, ET(30), which is based on the negative solvatochromism of pyridinium N-phenolate- betaine dye [25]. To understand the solvatochromic shift through a single parameter, ET(30) values are calculated for all studied solvents [19]. To the best of our knowledge, investigations of the ground- state and excited-state dipole moments of AF514 and AF532 fluorescent dyes employing solvatochromism have not been reported in the literature. The previous reports deal with the use of these fluorophores in biological applications like identification of proteins, FRET, SET, and imaging cells, etc. Thus, it would be important and interesting to obtain information about the geometry and excited state activities of the molecule. In this framework, the present article aims to explore the influence of the solvent medium of varying polarity on the ground and excited state dipole moments. The investigation concerns the influence of alcohols and general solvents on electronic spectra of AF514 and AF532 dyes and the determination of ground and excited state dipole moments. Various solvent correlation techniques such as Bilot-Kawski [26], Lippert-Mataga [27,28], Bakshiev [29], Kawski-Chamma- Viallet [30,31] and microscopic solvent polarity parameter 𝐸𝐸𝑇𝑇𝑁𝑁were also used to examine the experimental results. Also, the value of ground-state dipole moments is computed by using density functional theory, while singlet excited state by time- dependent density functional theory formalism. Computational solvatochromic analysis was carried out according to AM1/IEF- PCM and ZINDO/IEF-PCM for ground and excited states, respectively. Furthermore, natural bond orbital analysis was carried out to investigate the stabilization energy, hyper- conjugative interaction and charge delocalization within the studied molecules. The theoretically calculated dipole moments follow a similar trend with those estimated experimentally for both Alexa Fluor dyes. 2. Experimental Alexa Fluor 514 and Alexa Fluor 532 were purchased from Thermo Fisher Scientific (Life Technologies, USA) and used without any further purification. All solvents used for the study are of the highest spectroscopic grade, purchased from Sigma- Aldrich (HPLC grade). The absorption spectrum was measured using a UV-VIS- NIR spectrophotometer (JASCO, Model V-670) in the range of 300-700 nm. The PL spectra were recorded using a spectro- fluorometer (JY Horiba, Model Fluoromax4). A quartz cuvette with a path length of 1 cm was used with both the excitation and emission slit widths fixed at 1 nm with integration time 0.1 s/nm. Data analysis was performed using OriginPro 8.0 software. Ground state optimization and exited state studies were carried out by adopting DFT and TD-DFT at the level of CAM-B3LYP/6-311G(d,p) basis set and solvent effects were simulated by implementing AM1-IEFPCM/ZINDO-IEFPCM using the Gaussian16 package. 3. Theoretical background The ground and excited-state dipole moments of the solute provide information about the change in charge distribution and excited-state activities upon excitation. Several solvato- chromic correlation methods have been used for AF514 and AF532. 3.1. Bilot-Kawski method Bilot and Kawski [26] obtained a quantum mechanical relation using absorption (ῡa) and fluorescence (ῡf) band shifts measured in different solvents of varying permittivity (𝜀𝜀) and refractive index (n). Accordingly, the absorption (ῡa) and fluorescence (ῡf) maxima (in cm-1) can be expressed by solvatochromism equations. ( , )(1)v v m f na f B K ε −− − = − + constant (1) [ ( , ) 2 ( )](2)v v m f n g na f B K ε − − + = − +− + constant (2) 10 Patil et al. / European Journal of Chemistry 13 (1) (2022) 8-19 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.8-19.2123 where 2 22 1 1 1( , ) 2 222 2 n nf n n n εε ε  + − − =  −  + + +  and 43 1( ) 2 22 ( 2) ng n n  − =  +  are solvent polarity functions with the permittivity of solvents (ε ); refractive index (n), mB-K(1) and mB-K(2) are slopes obtained from the Equations (1) and (2), respectively, and are given below: 22( ) (1) 3 e gmB K hca µ µ− =− (3) and 2 22( ) (2) 3 e gmB K hca µ µ− =− (4) where c is the velocity of light in vacuum, ‘a’ the Onsager cavity radius of the solute, and h is the Planck’s constant. Considering that symmetry of the solute molecule remains unchanged upon electronic transition, then ground state (µg) and excited state (µe) dipole moments are parallel [14]. In such cases µg and µe are given by 1 2- 3(2) (1) 2 2 (1) m m hcaB K B K g mB K µ  − −  =   −  (5) 1 23(2) (1) 2 2 (2) m m hcaB K B K e mB K µ  +− −  =   −  (6) and (2) (1) (2) (1) m mB K B K e gm mB K B K µ µ +− −= −− − for mB-K(2) > mB-K(1) (7) Generally, the dipole moments (µg) and (µe) are not parallel to each other and make an angle φ between them, which can be estimated using Equation (8) [32] (1) (2) 1 2 2 2 2cos ( ) ( ) 2 B K e e B K m g gmg e φ µ µ µ µ µ µ − −   = + − −     (8) 3.2. Lippert-Mataga, Bakhshiev, and Kawski-Chamma- Viallet method Experimental ground and singlet excited state dipole moments can also be determined by the solvatochromic methods given by Lippert-Mataga Equation (9) [27,28], Bakhshiev Equation (10) [29] and Kawski-Chamma-Viallet Equation (11) [30,31], ( , )v v m F na f L M L M ε − − − = − − + Constant (9) ( , )v v m F na f B B ε − − − = + Constant (10) ( , ) 2 v va f m F nK C V K C V ε − − + = − − − − − + Constant (11) where, 22( ) 3 e gmL M hca µ µ− =− (12) 22( ) 3 e gmB hca µ µ− = (13) 2 22( ) 3 e gmK C V hca µ µ− =− − (14) mL-M, mB, mK-C-V are slopes produced from plots (ῡa -ῡf) vs FL- M, FB and (ῡa + ῡf)/2 vs FK-C-V and FL-M, FB, FK-C-V are solvent polarity functions given by Equations (15)-(17). 21 1( , ) 22 1 2 1 nF nL M n εε ε − − = −− + + (15) 2 22 1 1 1( , ) 2 22( 2) 2 n nF nB n n εε ε  + − − =  −  + + +  (16) 2 2 42 1 -1 -1 3( -1)( , ) -- - 2 2 2 222( 2) 2 2( 2) n n nF nK C V n n n εε ε   +  = +  + + + +   (17) 3.3. Empirical microscopic solvent polarity parameter The empirical microscopic solvent polarity parameter scale [ NET ] proposed by Reichardt [19] correlates better with the spectral shift of molecule than the traditionally used technique based on bulk solvent polarity functions. The correlation between the microscopic solvent polarity parameter ( NET ) with spectral shifts is given by Nv v m Ea f T − − − = + constant (18) with 2 3 11307.6 aBm aB µ µ     ∆  =     ∆      (19) where aB = 6.2 Å and ΔµB = 9 D are radius of Onsager cavity and change in ground and excited state dipole moment of Betaine dye on excitation ‘a’ and Δµ correspond to Alexa Fluor molecules; NET , the normalized solvent polarity function proposed by Reichardt [33] is a dimensionless solvatochromic parameter defined based on the absorption wave number ῡa of a standard Betaine dye in the solvent. ( ) ( ) ( ) 30.7 ( ) ( ) 32.4 E Solvent E TMS E SolventN T T TET E Water E TMST T − − = = − (20) where TMS represents tetramethylsilane, ET (solvent) empirical solvent polarity parameter ranges from 0 for TMS, to 1.000 for water extreme polar. Using these values, the change in dipole moments can be determined by Equation (21) 1/2 81 3(6.2/ ) 11307.6 m e g a µ µ µ  × ∆ = − =      (21) Patil et al. / European Journal of Chemistry 13 (1) (2022) 8-19 11 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.8-19.2123 Table 1. Spectral and photophysical parameters of AF514 and AF532 in various solvent polarity. Solvents λabs (nm) λemi (nm) 𝐯𝐯�𝒂𝒂, cm-1 𝐯𝐯�𝒇𝒇, cm-1 𝐯𝐯�𝒂𝒂 − 𝐯𝐯�𝒇𝒇, cm-1 𝐯𝐯�𝒂𝒂 + 𝐯𝐯�𝒇𝒇, cm-1 (𝐯𝐯�𝒂𝒂 + 𝐯𝐯�𝒇𝒇)/2, cm-1 AF514 AF532 Exp. Theo. * Exp. Theor. * AF514 AF532 AF514 AF532 AF514 AF532 AF514 AF532 AF514 AF532 AF514 AF532 Methanol 514 486 527 491 546 555 19455 18975 18315 18018 1140 957 37770 36993 18885 18496 Ethanol 517 488 528 493 553 552 19342 18939 18083 18115 1259 823 37425 37055 18712 18527 Butanol 518 492 531 492 541 561 19305 18832 18484 17825 820 1007 37789 36657 18894 18328 Hexanol 520 494 532 499 538 557 19230 18796 18587 17953 643 843 37818 36750 18909 18375 Decanol 521 499 532 503 539 558 19193 18796 18552 17921 640 875 37746 36718 18873 18359 Acetone 525 489 531 494 550 576 19047 18832 18181 17361 865 1471 37229 36193 18614 18096 Acetonitrile 519 487 526 503 542 558 19267 19011 18450 17921 817 1090 37718 36932 18859 18466 DMF 526 491 535 496 550 563 19011 18691 18181 17761 829 929 37193 36453 18596 18226 DMSO 529 490 539 495 551 568 18903 18552 18148 17605 754 947 37052 36158 18526 18079 Chloroform 528 504 531 508 550 549 18939 18832 18181 18214 757 617 37121 37047 18560 18523 * Theoretical values are estimated by Gaussian 16 With ZINDO/IEFPCM model. where m is the slope of the linear plot of NET vs. Stokes shift. 3.4. Onsager cavity radius The value of the Onsager cavity radius ‘a’ of AF514 and AF532 was determined by using Equation (22) [34,35] 1 33 4 Ma NAπρ    =    (22) where NA the Avogadro’s number, 𝜌𝜌 is the density and M molecular weight of the molecule. For studied molecules, a(AF514) = 5.034 Å and a(AF532) = 5.142 Å, respectively. 3.5. Computational studies The ground and excited state geometries of Alexa Fluor (AF514 and AF532) dyes were optimized using density functional theory and time-dependent density functional theory at the DFT/CAM-B3LYP/6-311G(d,p) and TD-DFT/CAM- B3LYP/6-311G(d,p) level in vacuum using Gaussian 16 software [36]. To explore the solvatochromism, the ground- state geometry of the fluorophores is optimized in all the solvents using semi-empirical method at the level AM1/IEF- PCM. ZINDO/IEF-PCM was used to obtain the transition energies and electronic absorption spectra in the solvent system. The ground and excited-state dipole moment vectors, HOMO, LUMO, and MEP maps are computed using DFT and TD- DFT, respectively. 4. Results and discussion 4.1. Solvent effect on the absorption and fluorescence spectra of AF514 and AF532 dyes Steady-state absorption and fluorescence spectra of Alexa Fluor dyes were obtained in various solvents with different solvent polarities. Figure 2 shows the typical absorption and fluorescence spectra of AF514 and AF532 in alcohol and general solvents, respectively. The effects of solvent can bring major changes in the intensity, shape and position of the absorption and fluorescence bands, which signify whether excited or ground state is more stabilized with the solvent [37,38]. Hence, these changes are the result of a specific interaction between the solute-solvent molecules. The absorption and PL maxima in nanometers, wavenumber in cm- 1, and shift in spectra of AF514 and AF532 in different solvents are listed in Table 1. All the calculated parameters f(ε,n), g(n), and f(ε,n)+2g(n), and some physical constants, Reichardt parameter NET are listed in Table 2. Table 1 discloses the spectral position of the AF514 and AF532 dyes in various solvents. It is noticed that the absorption maxima of both dyes lie between (514-529) nm and (526-539) nm and exhibit a small magnitude of shift (15 nm for AF514 and 13 nm for AF532, respectively) between the selected solvents. In polar solvents (Methanol to decanol), the absorption maximum shifts from 514 to 521 nm and 527 to 532 nm for AF514 and AF532, respectively, leading to a red-shift or bathochromic shift. However, the fluorescence maxima of these dyes lie between (538-553) nm and (549-576) nm and show a slightly higher shift (15 and 27 nm for AF514 and AF532, respectively) in AF532 as compared to its excitation spectra. This suggests that both the AF514 and AF532 dyes are more stabilized in the singlet excited state (S1) relative to the ground state (S2). Its means that the polarity of solvents has affected the ground-state energy of the molecule less as compared to the excited state. Consequently, the value for the ground-state dipole moment would be smaller compared to that of the excited state. The observed value of the Stokes shift varies from 1259 to 957 cm-1 (for AF514) and 957 to 617 cm-1 (for AF532) with the change in polarity of the solvents (Table 1). Varying Stokes shift with changing solvent polarity implies that solute solvent interactions are different in excited and ground states; indicating significant change in the ground state geometry of the dyes. 4.2. Estimation of dipole moments using solvatochromism of dyes In solvatochromism, the dielectric constant (𝜀𝜀) and the refractive index (n) of solvents play a vital role in the spectral shift (Stokes shift). It means that the spectral shift is due to the specific interactions between solute-solvent molecules. It might be influenced by solvent parameters like hydrogen bond donor (HBD), hydrogen bond acceptor (HBA) and solvent polarity. To determine the dipole moments of these dyes various solvatochromism shift methods are used. The ground state dipole moment (𝜇𝜇𝑒𝑒) of AF514 and AF532 dyes were determined by Bilot-Kawski correlation (Equation (1)). The value of singlet excited state dipole moments of these dyes was determined by the correlation between shift in the spectra and solvent polarity as given by Bilot-Kawski (Equation (2)), Lippert-Mataga Equation (9), Bakhshiev Equation (10), Kawski-Chamma- Viallet Equation (11) and Reichardt correlation Equation (18). For both dyes, linear regression was carried out and the resulting data were fit into a straight line, whose slopes (mB-K(1), mB-K(2), mL-M, mB, mK-C-V and mR), intercepts and correlation coefficients are listed in Table 3. It is noticed that some solvents deviated from the linear fit, possibly due to short range solute- solvent interactions. It was observed that the correlation coefficients (R2) of AF514 and AF532 are found to be in the range 0.811 to 0.979 and 0.798 to 0.991, respectively, which are achieved by using more solvents to obtain good linearity. 12 Patil et al. / European Journal of Chemistry 13 (1) (2022) 8-19 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.8-19.2123 Table 2. Solvent parameters and calculated values for solvent polarity functions. Solvent n ε α Π* β N TE ET(30) f(ε,n) g(n) f(ε,n)+2g(n) f L-M (ε,n) fB(ε,n) fK-C-V (ε,n) Methanol 1.329 33.70 0.98 0.60 0.66 0.762 55.4 0.857 0.224 1.305 0.3090 0.8574 0.6528 Ethanol 1.369 24.30 0.86 0.54 0.75 0.654 51.9 0.812 0.245 1.303 0.2856 0.8092 0.6557 Butanol 1.399 17.40 0.84 0.84 0.47 0.586 49.7 0.749 0.271 1.291 0.2633 0.7494 0.6458 Hexanol 1.418 13.00 0.80 0.40 0.80 0.559 48.8 0.686 0.284 1.254 0.2431 0.6860 0.6269 Decanol 1.437 8.00 0.70 0.45 0.70 0.525 47.7 0.553 0.297 1.146 0.2041 0.5527 0.5727 DMSO 1.359 47.24 0.08 0.71 0.76 0.355 42.2 0.928 0.222 1.372 0.3038 0.8771 0.6830 DMF 1.430 38.25 0.00 0.88 0.69 0.386 43.2 0.792 0.245 1.281 0.2753 0.8394 0.7114 Acetonitrile 1.344 36.64 0.19 0.75 0.40 0.460 45.6 0.861 0.235 1.330 0.3049 0.8610 0.6648 Acetone 1.479 21.01 0.00 0.71 0.43 0.444 45.1 0.839 0.292 1.423 0.2442 0.7522 0.6999 Chloroform 1.446 4.89 0.44 0.00 0.58 0.259 39.1 0.370 0.302 0.975 0.1482 0.3708 0.4876 Table 3. Linear plot data of AF514 and AF532 obtained from different correlation methods. Methods Slope Intercept Correlation coefficient (R2) No. of data point AF514 AF532 AF514 AF532 AF514 AF532 AF514 AF532 Bilot-Kawski MB-K(1) 145.39 803.25 705.29 321.69 0.862 0.826 04:10 07:10 Bilot-Kawski MB-K(2) 6385.27 2053.55 46101.35 39059.38 0.924 0.991 05:10 05:10 Lippert-Mataga mL-M 2772.75 2713.0 77.40 249.26 0.979 0.835 04:10 07:10 Bakhshiev mB 631.35 884.41 300.86 311.42 0.863 0.869 06:10 06:10 Kawski-Chamma-Viallet mK-C-V 5795.56 1218.50 22586.11 19104.12 0.842 0.887 06:10 05:10 Reichard mR 753.89 617.83 537.13 482.62 0.811 0.798 06:10 05:10 Figure 2. Normalized absorption and emission spectra of AF514 and AF532 in alcohols and general solvents. To determine the dipole moments experimentally, solvents correlation methods are used. The ground and excited state dipole moments ( c gµ and c eµ ) were calculated by using the slopes mB-K(1) and mB-K(2) of the Bilot-Kawski correlations using Equations (5) and (6). The values of the singlet excited state dipole moment were also determined from the slopes (mL-M, mB, mK-C-V and mR) of Lippert-Mataga, Bakshiv, Kawski-Chamma- Viallet and Reichardt correlations using Equations (15)-(17) and (21). The Onsager cavity radii of AF514 and AF532 are calculated using Edwards’ atomic increment method [35], and are tabulated in Table 4. Using Gaussian 16 software, the theoretical values of ground and singlet excited state dipole moments of AF514 and AF532 dyes were determined in vacuum. From Table 4, it is observed that the ground state dipole moments are found to be 16.15 D and 18.57 D while singlet excited state dipole moments are 16.61 D and 23.47 D, respectively. Also, it is noticed that for both molecules singlet excited state dipole moments (𝜇𝜇𝑒𝑒) are higher relative to ground state dipole moments as obtained from DFT and TDDFT computations as well as from Bilot- Kawski correlation method. This might be due to the significant redistribution of charge density between the electronic states, intramolecular bonding with solvents, charge transfer, and the nature of geometrical changes between the electronic states. This designates that both AF514 and AF532 dyes are significantly more polar in their excited state relative to the ground state. Therefore, both the studied molecules are more reactive in an excited state when interacting with the solvent. We have observed a comparatively good agreement between the excited state dipole moments determined by Bilot- Kawski, Lippert-Mataga, Bakhshiev, Kawski-Chamma-Viallet and Reichardt correlation methods. From Table 4, it can be seen that for both AF514 and AF532 dyes the excited state dipole moments obtained from the Lippert-Mataga method are higher as compared with the values obtained by other methods, since Patil et al. / European Journal of Chemistry 13 (1) (2022) 8-19 13 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.8-19.2123 Table 4. Ground and excited state dipole moments (‘μ’ in Debye), Onsager radius (‘a’ in Å) and angle between dipole moments (‘φ’ in degree) of AF514 and AF532 dyes. Compound 𝝁𝝁𝒈𝒈𝒂𝒂 𝝁𝝁𝒆𝒆𝒂𝒂 aj 𝝁𝝁𝒈𝒈𝒄𝒄 𝝁𝝁𝒆𝒆𝒄𝒄 𝝁𝝁𝒆𝒆𝒅𝒅 𝝁𝝁𝒆𝒆𝒆𝒆 𝝁𝝁𝒆𝒆 𝒇𝒇 𝝁𝝁𝒆𝒆 𝒈𝒈 𝝁𝝁𝒆𝒆𝒉𝒉 𝝁𝝁𝒆𝒆 /𝝁𝝁𝒈𝒈 ∅𝒊𝒊 AF514 16.15 16.61 5.034 29.13 30.49 35.05 31.95 30.36 30.83 1.70 1.35 0.50 AF532 18.57 23.47 5.142 2.56 5.86 8.62 6.02 4.80 4.15 1.59 3.29 0.49 a Computed at the DFT/CAM-B3LYP/6-311G(d,p) level using G16 software. b Computed at the TD-DFT/ CAM-B3LYP/6-311G(d,p) level using G16 software. c Calculated using the Bilot–Kawski method (Equations (1) and (2)). d Obtained using the Lippert Mataga method (Equation (9)). e Calculated using the Bakhshiev method (Equation (10)). f Obtained using the Kawski–Chamma–Viallet method (Equation (11)). g Obtained using the Reichardt method. h Difference in dipole moment. i Angle between dipole moments obtained using Equation (9). j Onsager radius obtained using Edward's atomic increment method. Table 5. List of experimental and computational results (solvent polarity scale ET(30) (in kcal/mol), transition energies (in eV), dipole moments (in D)). Solvents ET(30) EExp. ETheor. 𝝁𝝁𝒈𝒈𝒂𝒂 𝝁𝝁𝒈𝒈𝒃𝒃 ∆𝝁𝝁 𝝁𝝁𝒆𝒆/𝝁𝝁𝒈𝒈 Oscillator strength AF514 AF532 AF514 AF532 AF514 AF532 AF514 AF532 AF514 AF532 AF514 AF532 AF514 AF532 Methanol 55.4 2.412 2.353 2.549 2.529 20.585 23.985 30.670 32.622 10.085 8.637 1.490 1.360 0.8935 0.8817 Ethanol 51.9 2.398 2.348 2.536 2.511 20.514 23.910 30.561 32.515 10.047 8.605 1.490 1.360 0.8989 0.8874 Butanol 50.1 2.394 2.335 2.519 2.495 20.389 23.777 30.369 28.818 9.980 5.041 1.489 1.212 0.9060 0.9068 Hexanol 48.8 2.385 2.331 2.506 2.483 20.234 23.614 30.136 32.093 9.902 8.479 1.489 1.359 0.9112 0.9025 Decanol 47.7 2.380 2.331 2.483 2.462 19.898 23.249 29.625 31.576 9.727 8.327 1.489 1.358 0.9205 0.9160 Acetone 42.2 2.362 2.335 2.532 2.508 20.452 23.845 30.466 32.422 10.014 8.577 1.489 1.360 0.9003 0.8895 Acetonitrile 45.6 2.389 2.357 2.546 2.462 20.604 24.093 30.700 33.084 10.096 8.991 1.490 1.373 0.8947 0.8467 DMF 43.2 2.357 2.318 2.524 2.499 20.613 24.015 30.714 32.665 10.101 8.650 1.490 1.360 0.9032 0.8898 DMSO 45.1 2.344 2.301 2.530 2.504 20.655 22.092 30.779 32.727 10.124 10.635 1.490. 1.481 0.9008 0.8870 Chloroform 39.1 2.348 2.335 2.458 2.429 19.444 22.751 28.948 30.874 9.504 8.123 1.587 1.357 0.9296 0.9317 a Obtained employing AM1/IEF-PCM. b Obtained using ZINDO/IEF-PCM from G16 package. Figure 3. Optimized ground and excited state molecular geometries of AF514 and AF532 at the DFT/CAM-B3LYP/6-311G(d,p) and TD-DFT/CAM-B3LYP/6- 311G(d,p) level of theory in a vacuum. polarizabilities are not considered in the effect of the solute [39]. Using the Reichardt correlation method, the difference between 𝜇𝜇𝑔𝑔 and 𝜇𝜇𝑒𝑒were obtained by using Equation (21) and tabulated in Table 4. The ratio of singlet excited state and ground state dipole moments (𝜇𝜇𝑒𝑒/𝜇𝜇𝑔𝑔) were determined to be 1.35 and 3.29 for AF514 and AF532, respectively. The differ- ence in the dipole moment of the two electronic states is found to be positive and the ratio is also higher than unity for both dyes. The estimated value from AM1/IEF-PCM and ZINDO/IEF- PCM computations is analogous to the trend (Table 5) in all the solvents studied. The angle(𝜑𝜑) between the dipole moment vectors of two electronic states is calculated by using mB-K(1), mB- K(2), 𝜇𝜇𝑔𝑔, and 𝜇𝜇𝑒𝑒 obtained from the Bilot-Kawski correlation and were found to be 0.50 and 0.49° for AF514 and AF532, respectively, indicating dipole moments of the two electronic states to be parallel with each other. The parallelism of dipole moment vectors of the ground and excited states indicates a large charge movement through the molecule in both states. 4.3. Computational studies Quantum mechanical calculations were implemented to investigate the electronic properties and to gain enhanced insight information into the molecular structure of AF514 and AF532 dyes. The ground and excited state geometries are optimized using DFT and TD-DFT at the CAM-B3LYP/6- 311G(d,p) basis set, in a vacuum as shown in Figure 3. The arrowhead indicates the orientation of the dipole moment of the molecules. Further, the experimental transition energies of AF514 and AF532 are compared with the calculated ones used by ZINDO/IEF-PCM in different solvents (Figure 4). 14 Patil et al. / European Journal of Chemistry 13 (1) (2022) 8-19 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.8-19.2123 Figure 4. Comparison of experimental transition energies (eV) of AF514 and AF532 with computed by ZINDO-IEFPCM in different polarity of solvents. Figure 5. Molecular electrostatic potential (MEP) maps of AF514 and AF532 in gas phase. 4.3.1. Solvatochromism via ZINDO/IEF-PCM method The integral equation formalism polarizable continuum model (IEF-PCM) has been combined with the ZINDO semiempirical method to explain the effect of solvent on the geometry and electronic transitions of the molecule theore- tically. The solvatochromic effect on electronic absorption maxima, transition energy, and oscillating strength of AF514 and AF532 has been computed and analyzed by ZINDO/IEF- PCM in all the solvents studied. The ZINDO/IEF-PCM method is one of the most popular tools for the calculation of excitation energies compared with other semiempirical methods, as seen in the earlier reports [40,41]. In this paper, the ZINDO/IEF-PCM calculations of these fluorescent dyes are gathered in Table 5. It is noticed that the experimentally obtained data (shift 18 and 17 nm) of the absorption maxima of both dyes replicate the same trend as compared with the ZINDO/IEF-PCM data (shift 15 and 13 nm) in all kinds of solvents. The dependence of molecules on solvent polarity and solute-solvent interaction energy are studied along with experimental data. Figure 4 gives the typical plot of E (eV) vs. solvents polarities for AF514 and AF532; resulting data are collected in Table 5. In addition, the table discloses the maximum and minimum difference in the electronic transition energy of AF514 and AF532 in all solvents studied both experimentally and computationally. From experimental interpretation, the maximum and minimum energy differences of AF514 and AF532 are found to be 0.137- 0.103 eV and 0.176-0.131 eV in alcohols (methanol-decanol) and for general solvents (Acetone-Chloroform) and the computational study reveals them to be 0.186-0.110 eV and 0.203-0.094 eV, respectively. It can be noticed that the experimental and computed values are very close to each other. ZINDO/IEF-PCM examined electronic transition energies are well correlated with the experimental results with the minute differences of 0.137eV for AF514 and 0.176 eV for AF532 dyes. Conversely, the experimentally obtained results for the same are well-replicated for all the solvents. Oscillator strengths of AF514 and AF532 vary from 0.9205-0.8935 and 0.9160-0.8817 for alcohols and for other general solvents 0.9296-0.8947 and 0.9317-0.8467 as tabulated in Table 5. 4.3.2. Molecular electrostatic potential The molecular electrostatic potential (MEP) is a widely used tool to explain the reactive behavior of a variety of chemical systems in various environments, helpful for the study of biological recognition processes and hydrogen bonding interactions [42]. The MEP of AF514 and AF532 were calculated using DFT at CAM-B3LYP/6-311G (d.p) basis set as shown in Figure 5. The red (negative) region of the MEP map was associated with electrophilic reactivity and blue (positive) is associated with nucleophilic reactivity. The MEP maps are found to be in the region of -0.08407 (red) and 0.08407 (blue) for AF514 and -0.08376 (red) and 0.08376 (blue) for AF532. Note that the electron density map of AF514 has a much larger range than that of AF532. The mapped MEP surface (Figure 5) confirms that the red region on the oxygen atoms of the sulfonic groups is an electron rich (more electronegative) region for both molecules studied. Oxygen atoms attached with pyro- lidinyl are less electronegative (yellow) relative to sulfonic group atoms. Therefore, oxygen is relatively electron rich and hydrogen is relatively electron deficient for AF514 and AF532 molecules, respectively. 4.3.3 Frontier molecular orbitals The Frontier molecular orbital (FMO) analysis provides more useful information about the chemical reactivity and kinetic stability of the molecule. The forntier molecular orbitals called HOMO and LUMO of both studied molecules were investigated using DFT at the CAM-B3LYP/6-311G(d,p) basis set, in vacuum. Patil et al. / European Journal of Chemistry 13 (1) (2022) 8-19 15 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.8-19.2123 Figure 6. Frontier molecular orbitals of AF514 and AF532 in gas phase at CAM-B3LYP/6-311G (d,p) level. As observed in AF514, in HOMO electrons are restricted to benzopyran, quinoline, amine and sulfonic groups attached with quinolin, whereas in LUMO electrons are localized on all the groups except dioxopyrrolidinyl, 2-methyl groups and sulfonic acid groups. As is seen from HOMO-LUMO, electrons are delocalized on the oxygen atom of the benzopyran group and a phenyl group. In AF532, in HOMO electrons are restricted to pyran, indol, and sulfonic groups, whereas in LUMO electrons are localized in pyran, indol, and phenyl groups. In addition, electrons are delocalized in the oxygen atom of the pyran group and phyenyl group. For both the studied molecules electron density on the oxygen atom of benzopyran/pyran increases, at the same time electron density on oxygen atoms of sulfonic groups decreases during LUMO. The energies of the HOMO and LUMO can be directly related to ionization potential and electron affinity, respectively [43]. The HOMO-LUMO energy values were computationally deter- mined and revealed in Figure 6. For the dyes AF514 and AF532, HOMO-LUMO energy gap values were found to be 5.01 eV and 4.96 eV, respectively. The energy gap of AF532 is smaller than energy gap of AF514. This indicates that, the AF532 dye is highly reactive and has low kinetic stability as compared with AF514 dye. HOMO-LUMO energies are also used to find the softness (δ), chemical hardness (η), electron negativity (χ) and chemical potential (µ) of these molecules. For AF514 and AF532, the calculated values δ, η, χ, and µ are 0.399 eV-1, 2.505 eV, -4.985 eV, 4.985 eV and 0.403 eV-1, 2.480 eV, -4.850 eV, 4.850 eV, respectively. These values designate high excitation energy, high chemical activity, and good stability for the molecules studied. 4.3.4. Natural bond orbital (NBO) analysis The NBO analysis is an effective tool to study intra- and inter-molecular bonding and interactions. It also provides important features of the molecular structure and a basis for the investigation of charge transfer or conjugative interactions in molecular systems. This in turn offers insight into the intramolecular, intermolecular bonding, and interaction among bonds [44,45]. Another useful chemical aspect of the natural bond orbital method is that it gives a favorable interaction of both filled/partially filled orbitals with nearby empty/virtual orbitals, which explains certain chemical phenomena in terms of donor-to-acceptor orbital interactions [46]. The NBO analysis is carried out to understand various second-order interactions between the ‘filled’ (donor) Lewis type and ‘vacant’ (acceptor) non-Lewis type orbitals. NBO calculations were performed by using the NBO program as executed in the Gaussian 16 package at DFT/CAM-B3LYP/6- 311G(d,p) basis set. The second-order Fock matrix was carried out to determine the donor–acceptor interactions in the NBO analysis [47]. Second order perturbation theory is used for the filled donor NBOs (i) and vacant acceptor NBOs (j), the stabilization energy E(2) associated with i(donor) → j (acceptor) delocalization can be obtained as given by, 𝐸𝐸(2) = ∆𝐸𝐸𝑖𝑖𝑖𝑖 = 𝑞𝑞𝑖𝑖 (𝐹𝐹𝑖𝑖𝑖𝑖)2 (𝐸𝐸𝑖𝑖−𝐸𝐸𝑖𝑖) (23) where Fij is the off-diagonal NBO Fock matrix element between i and j, Ei and Ej are the energies of donor and acceptor orbitals of diagonal elements and qi is the occupancy of the donor orbital, ∆𝐸𝐸𝑖𝑖𝑖𝑖 is the difference in energy between the filled (donor) and the new lower energy orbital (formed by mixing of the donor and acceptor orbitals). In this NBO investigation, the higher E(2) value, the interaction between electron donors and acceptors is more intensive and the extent of conjugation is greater. The possible intensive perturbation energies of donor– acceptor interactions for AF514 and AF532 dyes are presented in Table 6. In AF532, the interactions between π*(C6-C7) and the antibonding acceptor π*(C8-C10) have a strong interaction with the highest intramolecular charge transfer energy E(2) value 284.13 kcal/mol. The lone pair electron donating 𝑛𝑛 → π* and 𝑛𝑛 → 𝜎𝜎*n1 transitions are observed between n1N19 to antibonding acceptor π*(C4-C5), n1N32 to π*(O30-C33), n2O12 to π*(C6-C7) and π*(C9-C12), n1O28 to 𝜎𝜎*(C26-C27), n2O29 to 𝜋𝜋*(C27- O28), and n2O30 to 𝜎𝜎*(N32-C33) and 𝜎𝜎*(C33-C35) transitions are responsible for resonance in the molecule with intramolecular charge transfer interaction energy E(2) value 86.27, 54.59, 40.95, 40.61, 20.44, 43.50, 37.86, and 24.64 kcal/mol, respectively. Although in AF514, the lone pair n→ π* transitions between n1C5 to π*(C4-C9) and π*(C6-C7), n1C11 to π*(C8-C10), π*(C12-C13) and π*(C15-C16), n1N19 to π*(O36-O38) and π*(O37- O39) transitions are significant interactions responsible for the resonance in the molecule with stabilization energies of 41.14, 57.54, 103.82, 92.36, 62.53, 54.31, 51.92, and 43.91 kcal/mol, respectively. Hence, the stabilization energies of AF532 is larger than stabilization energy of AF514. This indicates the more intramolecular charge transfers in AF532 as compared with AF514. i.e., AF532 is more stabilized relative to AF514. 16 Patil et al. / European Journal of Chemistry 13 (1) (2022) 8-19 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.8-19.2123 Table 6. Analysis of Fock matrix using second order perturbation theory for the compounds AF514 and AF532 dyes. Compound Donor Type ED/e Acceptor Type ED/e E(2)a E(j)-E(i)b F(i,j)c AF514 C4-C9 π 1.79133 C8-C10 π* 0.35249 16.92 0.38 0.074 C6-C7 π 1.73596 C8-C10 π* 0.35249 12.47 0.39 0.063 C8-C10 π 1.60720 C4-C9 π* 0.19437 25.19 0.40 0.095 C8-C10 π 1.60720 C6-C7 π* 0.37639 41.19 0.36 0.109 C27-C29 π 1.63579 C28-C30 π* 0.30184 25.83 0.37 0.089 - π - C31-C32 π* 0.35309 34.46 0.37 0.101 - π - C68-O70 π* 0.20239 14.50 0.41 0.072 C31-C32 π 1.63990 C27-C29 π* 0.38236 28.81 0.37 0.092 π - C28-C30 π* 0.30184 31.02 0.37 0.098 π - C33-O35 π* 0.14640 13.00 0.45 0.073 C5 n1* 0.86568 C4-C9 π* 0.19437 41.14 0.21 0.113 C5 n1* - C6-C7 π* 0.37639 57.54 0.16 0.108 C11 n1 1.10598 C8-C10 π* 0.35249 103.82 0.19 0.149 C11 n1 - C12-C13 π* 0.36417 92.36 0.19 0.138 C11 n1 - C15-C16 π* 0.20914 62.53 0.21 0.127 C14 n1 0.86526 C12-C13 π* 0.36417 58.66 0.16 0.112 C14 n1 - C15-C16 π* 0.20914 45.61 0.19 0.112 N19 n1 1.65778 O36-C38 π* 0.20780 54.31 0.41 0.138 N19 n1 - O37-C39 π* 0.20272 51.92 0.42 0.136 O20 n2 1.72369 C6-C7 π* 0.37639 43.91 0.45 0.129 O20 n2 - C12-C13 π* 0.36417 40.82 0.46 0.125 AF532 C6-C7 π* 0.37639 C8-C10 π* 0.35249 284.13 0.01 0.088 C3-C8 π 1.79416 C4-C5 π* 0.43630 25.65 0.37 0.093 - C6-C7 π* 0.47384 16.58 0.36 0.074 C4-C5 π 1.63755 C6-C7 π* 0.47384 48.25 0.35 0.119 - C3-C8 π* 0.20085 13.31 0.40 0.068 C6-C7 π 1.54132 C10-C11 π* 0.33650 40.33 0.36 0.112 - C3-C8 π* 0.20085 21.08 0.40 0.088 - C4-C5 π* 0.43630 15.21 0.35 0.066 C9-C12 π 1.70818 C10-C11 π* 0.33650 13.44 0.38 0.064 C10-C11 π 1.62847 C9-C12 π* 0.35870 36.73 0.37 0.105 - C14-C15 π* 0.18950 22.79 0.41 0.091 - C6-C7 π* 0.47384 15.63 0.35 0.068 C14-C15 π 1.80240 C10-C11 π* 0.33650 17.22 0.38 0.075 C21-C22 π 1.65230 C24-C26 π* 0.36271 33.80 0.37 0.100 - C23-C25 π* 0.28849 25.70 0.38 0.089 C23-C25 π 1.63839 C21-C22 π* 0.34730 35.16 0.36 0.101 - C24-C26 π* 0.36271 29.34 0.36 0.092 C24-C26 π 1.63839 C21-C22 π* 0.34730 28.15 0.36 0.091 - C23-C25 π* 0.28849 30.50 0.37 0.097 - C27-O28 π* 0.20068 23.94 0.37 0.087 C13 n1* 0.85834 C14-C15 π* 0.18958 44.47 0.20 0.116 - C9-C12 π* 0.35870 61.87 0.16 0.114 O12 n2 1.71637 C6-C7 π* 0.47384 40.95 0.44 0.126 - C9-C12 π* 0.47384 40.61 0.47 0.125 N19 n1 1.65100 C4-C5 π* 0.43630 86.27 0.34 0.157 O28 n2 1.83241 C26-C27 𝜎𝜎* 0.06309 20.44 0.82 0.119 O29 n2 1.84634 C27-O28 π* 0.20068 43.50 0.48 0.130 O30 n2 1.84677 N32-C33 𝜎𝜎* 0.10535 37.86 0.78 0.156 O30 n2 - C33-C35 𝜎𝜎* 0.06515 24.64 0.75 0.124 N32 n1 1.66192 O30-C33 π* 0.20182 54.59 0.41 0.138 a E(2) is the stabilization energy in kJ/mol. b Difference in energy (a.u.) between donor (i) and acceptor (j) NBO orbitals. c F(i,j) is the Fock matrix element (a.u.) of the NBO orbitals. Table 7. NBO results showing the formation of Lewis and non-Lewis orbital for Alexa Fluor 514 dye. Compound Bond (A–B) ED/energy (a.u.) EDA% EDB% NBO s % p % AF514 π C4-C9 1.79133 52.59 47.41 0.7252(sp99.99)C+ 0.01 99.92 π C6-C7 1.73596 61.25 38.75 0.7826(sp99.99)C+ 0.08 99.89 π C8-C10 1.60720 59.18 40.82 0.7693(sp1.00)C+ 0.01 99.97 π C12-C13 1.71092 40.70 59.30 0.6380(sp1.00)C+ 0.00 99.93 π C15-C16 1.80796 53.19 46.81 0.7293(sp1.00)C+ 0.00 99.94 π C27-C29 1.63579 47.70 52.30 0.6907(sp1.00)C+ 0.01 99.96 π C28-C30 1.62290 51.41 48.59 0.7170(sp1.00)C+ 0.00 99.95 π C31-C32 1.63990 46.13 53.87 0.6792(sp1.00)C+ 0.00 99.95 π C33-O35 1.98408 33.04 66.90 0.5748(sp27.14)C+ 3.54 96.00 π O36-C38 1.99033 68.90 31.10 0.8300(sp99.99)O+ 0.04 99.83 π O37-C39 1.98959 68.59 31.47 0.8278(sp99.99)O+ 0.22 99.65 n1*C5 0.86568 - - sp1.00 0.00 99.99 n1C5 1.10598 - - sp1.00 0.00 99.99 n1*C14 0.86526 - - sp1.00 0.00 100.00 n1N17 1.60446 - - sp99.99 0.17 99.81 n1N18 1.66606 - - sp99.99 0.04 99.94 n1N19 1.65776 - - sp99.99 0.68 99.93 n2O20 1.72369 - - sp1.00 0.01 99.93 Note: The symbols and labels appeared in the NBO analysis (Tables 6-8) were assigned according to the optimized structure as shown in Figure 4. Patil et al. / European Journal of Chemistry 13 (1) (2022) 8-19 17 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.8-19.2123 Table 8. NBO results showing the formation of Lewis and non-Lewis orbital for Alexa Fluor 532 dye. Compound Bond (A–B) ED/energy (a.u.) EDA% EDB% NBO s % p % AF532 πC3-C8 1.79416 52.03 47.97 0.7213(sp99.99)C+ 0.02 99.93 πC4-C5 1.63755 35.91 64.09 0.5993(sp1.00)C+ 0.00 99.93 πC6-C7 1.54132 40.49 59.51 0.6363(sp1.00)C+ 0.00 99.96 πC9-C12 1.70818 41.24 58.76 0.6422(sp1.00)C+ 0.00 99.93 πC10-C11 1.62847 58.58 41.42 0.7654(sp1.00)C+ 0.00 99.97 πC14-C15 1.80240 51.40 48.60 0.7170(sp99.99)C+ 0.03 99.91 πC21-C22 1.65230 50.41 49.59 0.7100(sp1.00)C+ 0.00 99.96 πC23-C25 1.63787 52.19 47.81 0.7224(sp1.00)C+ 0.00 99.95 πC24-C26 1.63839 45.29 54.71 0.6730(sp1.00)C+ 0.00 99.95 πC27-O28 1.98544 30.94 69.06 0.5562(sp1.00)C+ 0.00 99.46 πO30-C33 1.99107 68.67 31.33 0.8286(sp1.00)O+ 0.00 99.86 πO31-C34 1.99106 68.66 31.66 0.8286(sp1.00)O+ 0.00 99.86 n1*C13 0.85834 - - Sp99.99 0.02 99.98 n2O18 1.71637 - - Sp99.99 0.05 99.89 n1N19 1.65100 - - Sp63.07 1.56 98.41 n1N20 1.62467 - - Sp35.95 2.71 97.26 n2O28 1.83241 - - sp1.00 0.00 99.91 n2O29 1.84634 - - sp1.00 0.00 99.94 n2O30 1.84677 - - sp1.00 0.00 99.92 n1N32 1.66192 - - Sp99.99 0.37 99.62 n2O45 1.79558 - - sp1.00 0.00 99.91 n2O46 1.82894 - - Sp99.99 0.11 99.81 n2O47 1.90071 - - Sp99.99 0.15 99.80 n2O49 1.84079 - - sp1.00 0.01 99.92 n2O50 1.80945 - - sp1.00 0.00 99.91 a ED/e in a.u. Tables 7 and 8 gives the occupancy of electrons and p- character in the significant NBOs of the studied molecules [44]. In AF514, the 100% p-character was observed in the lone pair n1* C14. Except π bonding of (C33-O35) all the intense NBOs have the p-character very closer to 100%. Also, in AF532 except for lone pairs N19 and N20 all the significant NBOs have the p- character very closer to 100%. 5. Conclusion Herein we report, the effect of solvents on steady-state absorption and fluorescence spectra of AF514 and AF532 dyes in various solvents of differing polarities. These data are used to understand the solvatochromic effect on spectral shift; estimate the ground and singlet excited state dipole moments by using various correlation methods and to compare with computational studies. The ground state dipole moment is estimated by Bilot-Kawski’s method and singlet excited state dipole moments by employing Bilot-Kawski, Lippert-Mataga, Bakhshiev, Kawski-Chamma-Viallet and Reichardt’s methods. The dipole moments determined by using these correlation methods follow the same trend exhibited by theoretically calculated values using G16 software. Both experimental and computational results show that for these dyes singlet excited state dipole moment values are larger than those of ground state. This suggests that AF514 and AF532 are significantly more polar in the excited state than in their ground state. The ZINDO was adapted with integral equation formalism for the polarizable continuum model (IEF-PCM) to study the solvation potential and absorption transition energies. The experimental values of transition energy follow a similar trend exhibited by ZINDO/IEF-PCM model. The differences noticed in the transition energies are below 0.186 eV and 0.203 eV, respectively, in all the solvents. The small value of the HOMO- LUMO energy gap reveals the easy charge transfer interaction and softness of molecule. The intermolecular charge transfer between the bonding and antibonding orbitals and hybridization in the molecules under study has been understood by NBO analysis. It is observed that stabilization energies of AF532 are larger as compared with AF514 and it is more stable. Acknowledgment The authors would like to thank the University Grants Commission (UGC), New Delhi, India for the financial support under CPEPA (F.No.8-2/2008 (NS/PE)) and the USIC, Karnatak University, Dharwad for providing the UV- Vis Spectrophotometer. Gaussian calculations were performed in UPE-FAR-I Molecular Modelling Lab., Karnatak University, Dharwad. The author Mallikarjun would like to thank KSTePS Govt. of Karnataka for Providing DST- Ph.D. fellowship and Tarimakki Shankar Tilakraj is thankful to UGC for an SRF. Disclosure statement Conflict of interests: The authors declare that they have no conflict of interest. Ethical approval: All ethical guidelines have been adhered. Sample availability: Samples of the compounds are available from the author. CRediT authorship contribution statement Conceptualization: Sanjeev Ramchandra Inamdar, Mallikarjun Kalagouda Patil; Methodology: Mallikarjun Kalagouda Patil, Tarimakki Shankar Tilakraj, Mare Goudar Kotresh; Software: Mallikarjun Kalagouda Patil, Tarimakki Shankar Tilakraj; Validation: Mallikarjun Kalagouda Patil; Formal Analysis: Mallikarjun Kalagouda Patil, Tarimakki Shankar Tilakraj, Mare Goudar Kotresh; Investigation: Mallikarjun Kalagouda Patil, Tarimakki Shankar Tilakraj, Mare Goudar Kotresh, Sanjeev Ramchandra Inamdar; Resources: Mallikarjun Kalagouda Patil, Mare Goudar Kotresh; Data Curation: Mallikarjun Kalagouda Patil, Mare Goudar Kotresh; Writing - Original Draft: Mallikarjun Kalagouda Patil; Writing - Review and Editing: Mallikarjun Kalagouda Patil, Tarimakki Shankar Tilakraj, Mare Goudar Kotresh, Sanjeev Ramchandra Inamdar; Visualization: Mallikarjun Kalagouda Patil, Mare Goudar Kotresh, Sanjeev Ramchandra Inamdar; Funding acquisition: Sanjeev Ramchandra Inamdar; Supervision: Sanjeev Ramchandra Inamdar; Project Administration: Sanjeev Ramchandra Inamdar. ORCID and Email Mallikarjun Kalagouda Patil mkpatilphy@gmail.com https://orcid.org/0000-0001-7126-9486 Mare Goudar Kotresh kotreshm26@gmail.com https://orcid.org/0000-0001-8738-2221 Tarimakki Shankar Tilakraj tstilakraj1@gmail.com https://orcid.org/0000-0001-5719-3593 mailto:mkpatilphy@gmail.com https://orcid.org/0000-0001-7126-9486 mailto:kotreshm26@gmail.com https://orcid.org/0000-0001-8738-2221 mailto:tstilakraj1@gmail.com https://orcid.org/0000-0001-5719-3593 18 Patil et al. / European Journal of Chemistry 13 (1) (2022) 8-19 2022 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.13.1.8-19.2123 Sanjeev Ramchandra Inamdar him_lax3@yahoo.com https://orcid.org/0000-0003-3398-4897 References [1]. Patil, M. K.; Kotresh, M. G.; Inamdar, L. S.; Inamdar, S. R. Multidonor Surface Energy Transfer from Alexa Fluor Dyes to Gold Nanoparticles: A Quest for Innovative Sensor Applications. J. Nanophotonics 2020, 14 (03), 036006. [2]. Conroy, E. M.; Li, J. J.; Kim, H.; Algar, W. R. 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Copyright © 2022 by Authors. This work is published and licensed by Atlanta Publishing House LLC, Atlanta, GA, USA. The full terms of this license are available at http://www.eurjchem.com/index.php/eurjchem/pages/view/terms and incorporate the Creative Commons Attribution-Non Commercial (CC BY NC) (International, v4.0) License (http://creativecommons.org/licenses/by-nc/4.0). By accessing the work, you hereby accept the Terms. This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 3. Theoretical background 3.1. Bilot-Kawski method 3.2. Lippert-Mataga, Bakhshiev, and Kawski-Chamma-Viallet method 3.3. Empirical microscopic solvent polarity parameter 3.4. Onsager cavity radius 3.5. Computational studies 4. Results and discussion 4.1. Solvent effect on the absorption and fluorescence spectra of AF514 and AF532 dyes 4.2. Estimation of dipole moments using solvatochromism of dyes 4.3. Computational studies 4.3.1. Solvatochromism via ZINDO/IEF-PCM method 4.3.2. Molecular electrostatic potential 4.3.3 Frontier molecular orbitals 4.3.4. Natural bond orbital (NBO) analysis 5. Conclusion Acknowledgment Disclosure statement CRediT authorship contribution statement ORCID and Email References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField16: PrintField17: PrintField18: PrintField19: PrintField110: PrintField111: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: PrintField26: PrintField27: PrintField28: PrintField29: PrintField210: PrintField211: