Accepted Manuscript Additive-induced aggregate changes of two structurally similar dyes in aqueous solutions: A comparative photophysical study

A. Ghanadzadeh Gilani, Z. Poormohammadi-Ahandani, R. Kian PII: DOI: Reference:

S1386-1425(17)30678-9 doi: 10.1016/j.saa.2017.08.048 SAA 15405

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date: Revised date: Accepted date:

7 June 2017 1 August 2017 16 August 2017

Please cite this article as: A. Ghanadzadeh Gilani, Z. Poormohammadi-Ahandani, R. Kian , Additive-induced aggregate changes of two structurally similar dyes in aqueous solutions: A comparative photophysical study, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2017), doi: 10.1016/j.saa.2017.08.048

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Additive-induced aggregate changes of two structurally similar dyes in aqueous solutions: A comparative photophysical study

IP

T

A. Ghanadzadeh Gilani 1*, Z. Poormohammadi–Ahandani 1, R. Kian 2

1) Department of Chemistry, Faculty of Science, University of Guilan, Rasht, Iran

CE

PT

ED

M

AN

US

CR

2) Research Institute for Applied Physics and Astronomy, University of Tabriz, Tabriz, Iran

AC

* Corresponding author Address:

Prof. A. Ghanadzadeh Gilani, Department of Chemistry, University of Guilan, 41335 Rasht, Iran Tel: 0098 131 3233262, Fax: 0098 131 3233262 E-mail address: [email protected] (A. Ghanadzadeh Gilani)

1

AC

CE

PT

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

2

ACCEPTED MANUSCRIPT

Abstract

Absorption and emission spectral characteristics of the two structurally similar phenothiazine dyes, azure B and toluidine blue, in aqueous solutions of the two sets of

IP

T

molecular additives (ureas and monosaccharides) were studied as a function of the dye and

CR

additive concentrations. The absorption spectra of the dyes were also studied in pure tetramethylurea with an aprotic nature. The spectral data were analyzed using DECOM

US

Program. The dimer structure of the interacting molecules in these dyes was discussed using the exciton model. The urea class of additives was found to act as water structure-

AN

breakers over the range of studied concentration. The carbohydrate additives were found to

M

act as water structure-breakers at low concentrations. However, the water structure

ED

breaking process may be disfavored by the additive-additive interactions at higher concentrations. It can be concluded that at low additive concentrations, the main driving

PT

force for breaking the dye association is water-additive interaction, which disrupts the water hydrogen bonds induced by the additives. However, at the high additive

CE

concentrations, the different phenomena including additive–additive and additive-dye

AC

interactions can change the structure, strength, and aggregative properties of the dyes. Finally, the urea in water induces noticeably fluorescence quenching in emission spectra of both the dyes.

Keywords: Phenothiazines; Azure B; Toluidine blue; Dimerization; Additive effect

3

ACCEPTED MANUSCRIPT

1.

Introduction

Thiazines are among the most important organic dyes and colorants that have variety applications in science and technology [1]. They are used in various aspects of chemical,

IP

T

biological, and medical studies [2]. In recent years, many important efforts have been

CR

carried out on the photochemical and photophysical behavior of this class of dyes [35].

US

Dimerization and/or higher order aggregation of ionic dyes in solution is a well-known phenomenon [6]. The existence of the aggregate species plays a fundamental role in

AN

chemical and biological processes. In aqueous solution, phenothiazine dyes exhibit

M

aggregations mainly due to the hydrophobic character of the molecular structure. This

ED

process effectively depends on several factors including dye concentration, structure and

PT

type of solvents [79].

CE

The analysis of the spectral data and explanation of aggregate formation of phenothiazines in aqueous solutions have been performed by several researchers [1013]. However, the

AC

strength of dye association and spectral performance of this class of dyes strongly depends on the environment experience by the molecules or ions. The nature and structure of the dye aggregates have spectroscopically been investigated in aqueous environment under different conditions including presence of different types of additives, clays, and surfactants [1417]. Various parameters such as the charge and the alkyl tail length of the surfactants and the type and the position of the substituents in the aromatic ring of the dye molecules

4

ACCEPTED MANUSCRIPT

can affect the interactions between surfactant and dye molecules [18]. Dye-surfactant aggregates may occur in the premicellar region [19]. Electrostatic attraction or repulsion forces are much important than the other interaction forces in the ionic dye-surfactant

IP

T

systems [20].

CR

The presence of organic additives in water may highly change the dye solution properties because of the water-additive and dye-additive interactions [2124]. Molecular organic

US

additives with various concentrations have many applications in science and technology

AN

[2527]. They have a significant role in changing or improving the solution properties, as they strongly influence various phenomena in solutions such as solute-solute (self-

M

association and ion pairing) and solvent-solvent interactions in aqueous media. When

ED

molecular additives are added to the dye-water solutions, the dye molecules can interact with both the additive and water molecules. The additive can cause changes in structure and

PT

degree of the dye association through the water structure breaking or making properties

CE

[2830].

AC

Based on molecular structures, two groups of five types of organic additives were used in the current study (Fig. 1a). The first group of the molecular additives is composed of urea (U), tetramethylurea (TMU), and acetylenediurea (ADU). The second group composed of monosaccharides (carbohydrates), i.e. D(-)-fructose (Fru) and D-glucose (Glu). Both the ureas and carbohydrate molecules are the most fascinating additives that influence the water structure or water hydrogen bonds. Moreover, the choice of these two classes of

5

ACCEPTED MANUSCRIPT

additives is related to the interesting multifunctional structures, which can interact with water, themselves, and also with the dye molecules through the hydrogen bonding and dipole–dipole interactions. Consequently, a comparative spectroscopic study on the spectral and aggregative behavior of azure B (AB) and toluidine blue (TB), with strong similar

CR

IP

T

molecular structure, was carried out in this investigation.

Water molecules with up to four hydrogen bonds in a tetrahedral geometry are known to

US

have an extended hydrogen bond network structure [31]. Due to the stiffness and hydrogen bonded network of water, it is a highly ordered and associated liquid [32]. The extent of

AN

this network structure can be perturbed by the incorporation of dissolved solute or additive

M

molecule, and thus the structural evolution of the water–additive or water-solute network is

ED

different from the water-water hydrogen bond network [33]. The concept of the water structure breaking and making effect of solutes, which was first introduced by Gurney [34]

PT

can be explained in terms of altered water structure. Accordingly, structure breaking or making effect of additives (on the hydration water) has been used for interpreting additive-

CE

water interactions through changes in bulk solution properties. In general, structure

AC

breakers weaken the hydrogen-bonded structure of water and structure makers are used to enhance the stiffness of the water hydrogen bond network [35].

Urea (U) molecules are able to form hydrogen bonds with water molecules and also with themselves. It weakens hydrogen-bond structure of water and hydrophobic interactions [28]. Effect of urea on the hydrogen-bond structure of water has been the subject of continuing interest in recent years [3644]. The analysis of the various phenomena in the

6

ACCEPTED MANUSCRIPT

urea-water system has been reported in a review paper [45]. Tetramethylurea (TMU) belongs to the class of ureas but with unusual properties. It is a polar aprotic liquid that can participate in donor–acceptor H-bond formation with water molecules [46]. It uses as a reagent or intermediate in several chemical reactions [47]. Acetylenediurea (ADU) is

IP

T

composed of two urea group that poorly soluble in cold water. ADU and its derivatives are

CR

important chemicals for molecular, supramolecular, and combinatorial chemistry [48].

US

Carbohydrates with polar carbonyl and hydrophilic hydroxyl groups are very attractive additives that influence the water structure with the hydrogen bonding. D-(-) Fructose (Fru)

AN

and D-glucose (Glu) belong to monosaccharides, which include a carbonyl group (C=O).

M

However, these two organic compounds differ structurally. They are very soluble in water;

ED

however, the water solubility of Fru markedly is more than Glu [49]. They have little tendency to associate in aqueous solution [50]. Due to their specific structures including

CE

dye molecules as well.

PT

carbonyl and hydroxyl groups, both the hydrophilic compounds tend to interact with the

AC

The temperature and concentration effects of Fru on water structure have been recently reported [51]. According to this report, the hydrogen bonding capability of the -OH groups in Fru is mainly responsible for water–Fru interactions. Moreover, it has also been reported that Fru at lower concentrations acts as a water structure-breaker additive, whereas at higher concentrations it acts as a structure-maker additive. This effect might attribute to the less water–Fru interactions through H-bonding at lower concentrations and vice versa.

7

ACCEPTED MANUSCRIPT

As continuation of our previous spectroscopic studies on the effect of molecular additives on the photophysical and association properties of a range of ionic dyes [5255], we focus in this study on the two phenothiazine dyes, i.e. azure B and toluidine blue.

IP

T

In this report, we describe the effect of the selected molecular additives on the spectral and

CR

aggregative properties of the two phenothiazine dyes (Fig. 1b) in aqueous solutions. The structure of the dyes consists of an electron donor and an electron withdrawing aromatic

US

system. Both the dyes have hydrogen bond donor/acceptor groups, which form hydrogen bonds with water molecules or media containing molecules with hydrogen bond

AN

donor/acceptor ability. The additive molecules can form hydrogen bonds with water, dye,

M

and themselves. Fluorescence spectra of the dyes in the aqueous additive solutions were

ED

recorded and studied as well, in order to gain more information regarding the dye

Experimental

2.1

Materials

AC

CE

2.

PT

association behavior as a consequence of the additive induced by the additives.

The studied phenothiazine dyes (Azure B, and toluidine blue) were of analytical reagent grade and were obtained from Sigma-Aldrich. The molecular additives used in the current study; Urea (purity > 0.995), tetramethylurea (purity > 0.99), and acetylenediurea (purity > 0.98) were obtained from Merck, Aldrich, and Sigma-Aldrich respectively. D-Glucose (purity > 0.99) and D(-)-fructose (purity > 0.99) were supplied from Merck and SigmaAldrich, respectively.

8

ACCEPTED MANUSCRIPT

The chemicals were used directly as supplied commercially without any further purification. Distilled and deionized water with an electrical conductivity less than 5 S cm  was employed for the preparation of all solutions. The molecular structures of the

T

1

2.2

CR

IP

chemical used in this study are shown in Fig. 1(a, b) for a quick visualization.

Sample preparation

US

Stock solutions of each dye ( 5  10 4 M) were separately prepared by mass and dissolved in

AN

an appropriate amount of water in 100 ml volumetric flask at room temperature. A range of concentrations of the dye in aqueous solutions of the additives (the testing solutions) were

M

prepared using a Brand Transferpette micropipette and aliquot of the stock solutions The

ED

additive concentrations, except for acetylenediurea, were prepared up to 3 M. As the water solubility of acetylenediurea is small at room temperature, it was studied up to a

PT

concentration range of 0.01 M. The dye solutions were also prepared and examined in pure

CE

liquid tetramethylurea liquid as well as its aqueous solutions. The sample weighing was carried out using an electronic analytical balance (AND model HR-200) with an accuracy

AC

of ±0.1 mg.

2.3

Measurement of absorption and fluorescence spectra

A Cary UV–Vis double-beam spectrophotometer was used to record the absorption spectra of the studied dyes over a wavelength range between 350 and 800 nm at 25 o C . The sample holders were a series of optical cell with 1–10 mm path lengths. Precise values of max and

9

ACCEPTED MANUSCRIPT

shoulderwere obtained from the first derivative of the absorption spectra [56]. The uncertainty in the measured wavelength of absorption maxima was ± 0.1 nm. Fluorescence spectra of the dyes in aqueous and aqueous solutions of the additives were recorded on a

T

Shimadzu RF5000 spectrofluorimeter at room temperature. The fluorescence spectra were

CR

IP

recorded in dilute dye solution and were checked, in order to avoid self-absorption process.

Theoretical parts

2.4.1

Determination of the dimerization constant

AN

US

2.4

Assuming that only a single equilibrium between monomer (M) and dimer (D) exists, the

M

dimer association constant ( K d  [ D] /[ M ]2 ) can be determined from the spectral data and

ED

the following brief procedure. Detail description of the procedure has been proposed and introduced in our previous publication [57]. The total concentration (C) and total absorption

PT

( At ) can be defined as [C ]  [M ]  2[ D] and A total  A M  A D , respectively. The At can be

CE

express as A t  ε M M l  ε D D l . Where  M and  D correspond to molar absorptivities

length.

AC

of monomer and dimer, respectively, at a given wavelength (  ), and “ l ” is the optical path

For the calculation of the K d values of the dyes, the linear and nonlinear least squares fitting approaches were used. The fitting consist of two elements containing the dimerization constant calculation and spectra decomposition.

10

ACCEPTED MANUSCRIPT

2.4.1.1 Linear least squares fitting Dividing A t  ε M M l  ε D D l by [C ]  l and defining (   [M ] /[C ]  as the fraction of dye molecules present as monomeric form, the dimerization constant can be express as [54] K d  (1   )[C ] / 2 2 [C ]2

gives a straight line with intercepts at  M

CR

Variation of  t vs. 

IP

 t   M     D  (1   ) / 2   M   D / 2     D / 2

T

(1) (2)

for   1 and

US

 D / 2 for   0 . Therefore, the monomer and dimer spectra as well as optimum K d values

2.4.1.2 Non-linear least squares fitting

M

and [C ]  [M ]  2[ D] into A t  ε M M l  ε D D l , the

ED

Substituting K d  [ D] /[ M ]2

AN

can be obtained by analyzing the straight-line fitness in a series of wavelengths.

following equation was obtained [54]

At   M [1  1  8  K d  [C ] / 8  K d ]  l   D {[4  K d [C]  [1  (1  8  K d [C ]1/ 2 )]] / 8  K d }  l

(3)

PT

12

CE

Fitting the experimental data to the above equation (Eq. 3) and using nonlinear curve fitting in a series of wavelengths, the monomer and dimer spectra as well as optimum K d values,

AC

can be simultaneously determined.

2.4.1.3 The implementation of the linear and nonlinear approaches The performance of these methods was packed as an Add-in, i.e. Decom [57]. The data for “Decom” linear and nonlinear methods were determined through single step run over all range of log K d (0–15). Typically, the K d estimation process for one of the studied dyes,

11

ACCEPTED MANUSCRIPT

i.e. azure B, is shown in Supplementary data Fig. 1a. For the linear approach, the efficiency of linear fit for molar absorptivity vs.  (the fraction of dye molecules exist in monomeric form) is estimated (Supplementary data Fig. 1b). In the nonlinear method, the fit efficiency is defined as similarity between experimental and theoretical spectra (Supplementary data

IP

T

Fig. 1c). The most trustful results are those for which both the methods give similar data. It

CR

is worth to say, the program chooses the optimum K d value and decomposes the

Determination of the excitonic parameters

AN

2.4.2

US

absorption spectra.

Through the framework of the molecular exciton theory developed by Kasha et al. [58], the

M

character and structure of the interacting molecules in the dimeric units were obtained and

ED

discussed. The two extreme H- and J-dimer structures (parallel and linear structures) can be formed according to this model. They may be specified by the angle and distance between

CE

PT

the transition dipoles [59].

For real systems, both the parallel and linear structures can be simultaneously formed.

AC

Consequently, the intermediate dimeric structure result in the oblique type-packing should be considered as existence of J- and H- type dimers, which lead to appearance of both Hand J-bands placed at both side of the monomer band. Dimerization process caused by the dipole-dipole coupling between monomeric units splits the excited state level into two levels, while the ground state level of the dimer remains degenerated (Fig. 3). However, it has already been stated that splitting of both the ground and excited states may take place

12

ACCEPTED MANUSCRIPT

simultaneously [60]. The difference in energy of the split levels depends on the interaction energy (U) between the adjacent chromophores, which can be determined by considering the half of the frequency difference existing between the H-band and J-band maxima

1  H  J  2

IP

U (cm 1 ) 

T

( H and J ) in dimer spectrum [3,54]. (4)

CR

The angles existing between the chromophores (  ) and the transition moments (  ) can be

US

determined by the Eq. 5a and b, respectively [3]

  2 arctan  J /  H

AN

 H  fJ  J  fH

(5b)

M

  2 arctan

(5a)

ED

In which f H and f J are the oscillator strengths of the corresponding transition bands. The oscillator strengths ( f ) of the monomer and the dimer species were calculated using

PT

( f  4.32  109   ( ) d ). Here  ( ) is the extinction coefficient and  is in wave

AC

[3]:

CE

numbers. The distance between the monomeric units in the dimer can be estimated using

R3

2.14  1010  cos   f M   M

(6)

here f M and  M are the oscillator strength and energy of the transition in the monomer spectrum. The corresponding transition dipole moment (  ) can be calculated using [54]:

  2

e 2 3hf 8 2 m

13

(7)

ACCEPTED MANUSCRIPT where m and e are the mass of and charge on the electron, h Planck’s constant and  the frequency at maximum absorbance.

Results and discussion

3.1

Absorption spectral data

CR

IP

T

3.

In this research, the two sets of organic additives were proposed, which can alter water

US

structure ordering appearing as the dye spectral changes. The first set containing the ureas

M

monosaccharides (fructose and glucose).

AN

(urea, tetramethylurea, and acetylenediurea), and the second one was belong to

ED

The dye dimerization reduction induced by water structure change was studied through a series of absorption experiments under varying amount of the dye and the additive

PT

concentrations. For each dye-additive system, the test solutions were separately prepared. The absorption spectra of the phenothiazine dyes in aqueous solutions containing additives

CE

at 12 different dye concentrations ranging from 5  106 to 5  10 4 M were recorded at

AC

25 o C . The spectral data are summarized in Table 1.

Typically, Supplementary data Fig. 2 (a,b) shows the absorption spectra of AB and TB in aqueous solutions of ADU (0.01 M), at different dye concentration range. From the spectral feature, the shoulder on the short-wavelength side is developing, which can be attributed to the dimer formation.

14

ACCEPTED MANUSCRIPT

3.1.1

Ureas-induced spectra changes

A comparison of the spectral characteristics of AB and TB in presence of the molecular additives (ureas) with similar chemical functionality (amino and carbonyl groups) but

IP

T

different solubility and structure breaking properties was carried out. The molecular

CR

structures of this group of additives were containing primary (urea, U), secondary (acetylenediurea, ADU), and tertiary (tetramethylurea, TMU) amino groups. In our

US

previous publication [55], the thiourea-induced association change on these dyes was

AN

studied at room temperature.

M

The absorption spectra of the dyes in aqueous solutions of U (03M), at a fixed dye

ED

concentration ( Cdye  5  104 M) are shown in Fig. 3 (a,b). The dye spectra in water without urea at the same concentration are also presented for comparison. It can be observed that

PT

the spectral features markedly change by the increasing of urea concentration. The spectra

CE

show that extent of the dye aggregation decreases in aqueous solutions of urea. This process could be due to the water structure breaking or disruption of water structure

AC

induced by intermolecular interaction between urea and water or the dye molecules. However, at higher urea concentrations, the urea-urea interaction should be considered, which may disfavor the water structure breaking process [61].

The influence of the ureas addition and their chemical nature on the spectral performance of the dyes in water at the fixed dye ( 1 104 M ) and additive ( 1.6 M ) concentrations was

15

ACCEPTED MANUSCRIPT

analyzed and the result presented in Fig. 4 (a,b). The spectra of aqueous solution of ADU (0.01M) are not shown in this figure but it is overlapped with that of the TMU. The spectrum of each dye in pure water is also plotted for comparison. The observed effect (different strength characteristics) on the spectra can be attributed to the change in water

IP

T

structure induced by the various additive molecules. The difference in intermolecular

CR

interaction between the additive, water, and the dye molecules, suggest the different hydration shell structure around the dye molecules. The figure shows that the tendency of

AN

difference in degree was noted in theses cases.

US

the dye self-associations was reduced by the molecular additives. However, some

M

From the figure, the red-shift of the monomer absorption band with the increasing of the

ED

thiourea concentration is more evident than that of the other additives, which is indicating stronger hydrogen bond formation, as already reported by us [55]. The additive-induced

PT

red-shift is however, small compared to thiourea effect (8-9 nm). The magnitude of the expected spectral red-shift induced by the used additives was about 3-4 nm. It is worth to

CE

mention, in the high additive concentration range, the additive molecules in water are able

3.1.2

AC

to form aggregates through the intermolecular hydrogen bonds.

Monosaccharides-induced spectra changes

Absorption spectra of the aqueous dye solutions in the presence of glucose (Glu) or fructose (Fru) exhibit different characteristics compared to the dye spectra in pure water. Figs. 5 (ad) illustrate the absorption bands of the dyes in presence of Glu and Fru at a fixed dye

16

ACCEPTED MANUSCRIPT

concentration ( Cdye  5  10-4 M). The spectrum of each dye in water at the same concentration plotted as dashed line.

As can be seen, upon increasing Glu or Fru

concentration, the intensity of monomer absorption band of each dye increases in the

T

longer-wavelength side. It is believed that the strength of hydrogen bonded network of

CR

IP

water decreases due to an increase in the number of free water molecules.

Glu and Fru can change the water structure through water–additive interaction at the low

US

additive concentrations. However, this effect is not strong and they can act as relatively

AN

weak hydrogen bond breaker. These additives can also form hydrogen bonds directly with the amino groups of the dye molecules in competition with hydrating water molecules. In

M

addition, the Glu-Glu or Fru-Fru interaction through hydrogen bonding may influence

ED

considerably the water structure and the dye association. This observation can be realized using the idea those monosaccharides have different effects on dissociation and association

PT

behavior of the dyes at low and high concentrations. These monosaccharide additives, in

CE

particular Fru, can prevent or at least reduce the dye dissociation at the high concentrations.

AC

In other words, the water structure breaking process may be disfavored by the additiveadditive interactions. In the case of Fru-water system, our results confirm, at least partially, the main conclusion of a recently published paper. According to Afrin et al. [51], Fru behaves as water structure-breaker at low concentrations and at high concentrations act as structure-maker. However, from analysis of our spectral data, Glu or Fru act as reducer in the dye dissociation process at the high concentrations.

17

ACCEPTED MANUSCRIPT

3.2

The resolved monomer and dimer spectra

Fig 6 demonstrates the calculated monomer and dimer spectra of AB and TB in water and in the presence of the additives ([additive] =1.2 M). Due to the low solubility of ADU in

IP

T

water, its aqueous solutions were prepared up to 0.01 M. For compassion, the monomer

CR

and dimer bands of the dyes in pure TMU solvent are also demonstrated in Supplementary data Fig. 3. In spite of the spectral similarity of the dyes in aqueous and aqueous additive

US

solutions, their spectral and excitonic parameters are dissimilar. It can be seen that the intensity of H-band is higher than that of J-band, indicating a higher contributions of H-

AN

aggregate in the dye dimer structure. This is a consequence of the flat aromatic structures of

M

the dyes.

ED

However, urea and thiourea develop the (  J /  H ) values of the dyes in the additive

PT

concentration range; but, there is difference in degree between them. The literature data for

CE

water-TU system containing AB and TB were taken from our previous publication [55]. As can be observed from Fig. 7(a), on the addition of U or TU, the J- to H-band intensity ratio

AC

increases. Due to presence of the ureas additives in aqueous dye solutions, the simultaneous reduction of the dimerization process may takes place as well. In other words, they may accelerate formation of J-dimers. Similar trend has been reported for other phenothiazine dyes such methylene blue (MB) [10,55]. As can be seen from Table 1, The J- to H-band intensity ratio in the presence of Glu or Fru remains unchanged.

18

ACCEPTED MANUSCRIPT

Variation of the dissociation process in aqueous solution of ureas may be interpreted in terms of various inter species interactions between the dye, the additive, and water molecules. Its worth to say, this process is obvious for all the additives used in our current work but with different trends. The dissociation process for the studied dyes in aqueous

IP

T

solutions of ureas and monosaccharides, due to their water structure breaking ability, is in

CR

the following order, TU  U  Fru  Glu  TMU  ADU (0.01M) .

US

It can be concluded that TU is a relatively strong water structure breaker, TMU is a very weak hydrogen bond breaker, and urea can act as a moderate water-structure breaker. The

AN

results also show that Fru and Glu behave also as relatively weak water structure breaker at

3.3

ED

M

low concentrations.

The dimerization constants and excitonic parameters

PT

The excitonic parameters (U, R ,  K d ) of AB and TB dimers in aqueous and aqueous

CE

additive solutions were determined using the excitonic treatment, as discussed earlier. The calculated data are summarized in Table 1. Based on the close chemical and structural

AC

similarity between AB and TB, their calculated excitonic parameters in aqueous media are close. The variation of U, R , and  with increasing urea concentration is plotted in Fig. 7(bd). For both the dyes, the angles between the interacting molecules are relatively small (50o), which means that the dyes can form H-dimer by close packing of two neighboring molecules.

19

ACCEPTED MANUSCRIPT

A value of 1121 cm 1 was obtained for interaction energy ( U ) of AB in water. The U value is more than 1367 cm 1 obtained in this work for TB in aqueous solution. The interaction between the dye molecules in the dimers should have different origins. The

T

hydrophobic, dipole–dipole, and van der Waals forces (steric and dispersive) should have

IP

main contributions in the molecular arrangement of the dye in the dimer species. As is

CR

evident from Table 1, both the U and values decrease as the urea concentration increases, which is more evidence for AB. This might be attributed to water-structure breaking

US

properties of urea and its ability to change the dimer geometry from the parallel to linear

AN

structure. For both the dyes, the distance between the interacting molecules in the dimer (R), increases with increasing of urea concentration. As the R values depend on the dye-dye

M

separation, the distance between the neighboring monomers in the linear structure (J-dimer)

ED

is more than the parallel arrangement (H-dimer). Therefore, the increasing of the R values can be realized as urea has ability to change the H-type to J-type dimer. However, the

PT

change the dimer geometry in urea is week regarding to that of thiourea. The observed

CE

trend is not consistent in the case of Glu and Fru, as they are week structure breakers and

AC

very wake H-dimer to J-dimer switchers.

However, the influence of the attached groups in the dye aromatic skeleton is important in determining the arrangement of dye molecules in dimeric units. As can be seen from Table 1, there is some dissimilarity between their excitonic parameters, in particular, for the K d values. The K d values of AB in different media are higher than that of TB, which could be attributed to the higher hydrophobicity of AB. The additive concentration effect on the K d

20

ACCEPTED MANUSCRIPT

values of the dyes is shown in Fig 8 (a,b). As can be seen the degree of dimerization decreases in the aqueous additive solutions. Definitely, several factors have involved in some extent in this phenomenon. The water-additive, dye-additive, and additive-additive (at high concentrations) interactions have important contributions in the spectral and the dye

CR

IP

T

association behavior in the aqueous additive solutions.

The different trends for variation of K d observed in Fig. 8 for the dye association behavior

US

in presence of the additives can be due to different interactions between the molecular

AN

additives and the dye or water molecules. Fig. 9(ad) shows the different kind of intermolecular interactions that might be produced between the constituent molecules.

M

The used additives act as structure breakers in water at least at the low concentrations. They

ED

may modify the structure of solvation shell by replacement of water molecules in the hydration spheres of the dyes. However, the sharp decreasing trends are more evidence up

PT

to the concentration of 0.8 M. Above this concentration the interpretation is more

CE

complicated as different phenomena can simultaneously happened. It seems that urea

3.3

AC

behaves as a structure breaker additive in the range of concentration studied.

Absorption spectra of the dyes in pure tetramethylurea (TMU)

Effect of an organic polar aprotic solvent (TMU) on spectral and association behavior of the studied dyes, at various dye concentrations, was studied and compared with that of water. This liquid solvent has a moderate permittivity (   23.5 ), low viscosity (0.0140

21

ACCEPTED MANUSCRIPT poise), but relatively high molecular dipole moment (   3.7 D) [62]. Nevertheless, the association process between TMU-TMU molecules is not strong [63].

The absorption spectra, in terms of molar absorptivity, of AB and TB dissolved in pure

IP

T

TMU at different dye concentrations were recorded and is shown in Fig. 10 (a, b). For each

CR

dye, the intensity of monomer absorption band decreases and the intensity of the second band (shoulder) increases. Both the spectra recorded for AB and TB pass through isosbestic

US

points at 635 and 602 nm, respectively, indicating the presence of monomer–dimer equilibrium. The spectral parameters are given in Table 1. The dipole-dipole interaction is

M

AN

responsible for the dye aggregations in this polar aprotic solvent.

However, the dimerization process can be reduced through the hydrogen bonding between

ED

the dye and TMU molecules (Supplementary data Fig. 4). In pure TMU solvent, the

PT

tendency of self-association of both the dyes markedly decreases with that of that water.

CE

For AB and TB in water the dimerization constants ( K d ) were found to be 4898

M 1 and 3548 M 1 , respectively, which are close to previously reported data (i.e.

The

Kd

AC

4675 and 3311 M 1 ) [55] within the combined uncertainties of both the data sets. values for AB and TB in pure TMU solvent were estimated to be 1556, and 1

1236 M . The spectral changes indicate TMU-induced modifications of the dye aggregate behavior. This change is due to the fact that TMU can interact with the dye molecules at various sites via specific and nonspecific interactions. It is worth to say, the association process for the AB molecules in TMU as well as water is more than that of TB.

22

ACCEPTED MANUSCRIPT

3.4

Fluorescence spectra

The absorption and fluorescence spectra of the dyes in aqueous and aqueous additive solutions are shown in Fig. 11 (a,b). Both the dyes display single fluorescence bands in

IP

T

water in the presence of the molecular additives and the emission spectra maxima of the

CR

dyes are affected by their media. As can be seen from the figure, the fluorescence spectra of the dyes in the aqueous additive solutions were red shifted compared to pure water. In order

US

to avoid fluorescence self-absorption effect, the experiments were carried out at the low dye

AN

concentrations ( Cdye  5  106 M). As the fluorescence spectra of the dyes in aqueous and the aqueous additive solutions were recorded in dilute dye solutions, it can be assumed that

M

the dye molecules exist nearly in their monomeric form at the low concentrations.

ED

Therefore, the quenching is not through the dye-dye interactions.

PT

Fig. 12(a, b) shows the emission spectra of the dyes ( 5  106 M) in the aqueous and

CE

aqueous urea solutions at the two additive concentrations of 0.8 and 1.6 M. As it can be observed, the urea molecules in water induce markedly fluorescence quenching in emission

AC

spectra of the dyes. The quenching was larger at high urea concentration. Different factors may be involved in this process; however, the quenching through the dye-additive interactions can be more sensible.

The Stokes shift values for the studied dyes in the aqueous solutions of the molecular additives ([additive] = 0.8 M) are summarized in Table 2. It should be noted that the Stokes

23

ACCEPTED MANUSCRIPT

shift is the difference between the absorption and fluorescence spectra maxima of the molecules. In aqueous solution of the molecular additives, the Stokes shifts observed for the dyes were larger, which indicate that the dyes absorb light and emits fluorescence with

IP

T

larger rearrangement.

CR

The absorption and fluorescence spectra of the dyes in pure tetramethylurea as a dipole aprotic solvent are shown in Fig. 13 (a, b). The Stokes shifts for AB and TB were estimated

US

to be 449 and 680 cm 1 , respectively. TMU as a polar aprotic liquid is belongs to the class of ureas but with odd behavior. This liquid potentially has capability for specific

AN

interactions [64]. The polarity and hydrogen bonding properties of a solvent can be

M

characterized by the solvatochromic parameters α, β, and π* [62].  is the scale of solvent

ED

H-bond donor ability,  is the scale of solvent H-bond acceptor ability, and   is a measure of solvent dipolarity/polarizability. The solvatochromic parameters (  ,  ,  * ) of

PT

TMU are 0.00, 0.88, and 0.83, respectively. Thus, TMU is a good hydrogen bond acceptor,

Conclusions

AC

4.

CE

but is not a hydrogen bond donor.

It was shown that the self-association of the phenothiazine dyes, azure B and toluidine blue, in aqueous solution effectively decreases in the presence of the molecular additives. The hetero-associations of the dyes or water with the additive molecules results in decrease the dye dimerization. This process depends on the types of the molecular additives (ureas and monosaccharides). The dissociation process of the dyes in water is in the order of,

24

ACCEPTED MANUSCRIPT

TU  U  Fru  Glu  TMU  ADU . It may be mentioned here that the additive molecules induced changes in the structure and strength of hydrogen bonds in aqueous solution.

The excitonic parameters of the dyes in aqueous and aqueous additive solutions were

IP

T

evaluated and compared. In spite of their structural similarity, the excitonic parameters, in

CR

particular dimer association constant, K d , in aqueous solutions of urea are noticeably different. This difference may arises due to the presence of a –NH2 group attached to the

US

aromatic skeleton of toluidine blue, which make it more hydrophilic. The dyes form H-

AN

dimer by close packing with a small deviation from a parallel arrangement.

M

From the spectral data, there is an increase of J-dimer at the high urea concentrations. The

ED

 J /  H  1 in aqueous solution of urea in at a concentration of 3 M and 25o C specify that both the H-type and J-type dimers can exist in the aqueous solution of urea. However, there

PT

is a stronger tendency toward the formation of J-dimer, which was observed in an aqueous

CE

solution of thiourea. In contrast, no tendency to switch the dimer geometry from H-type to J-type dimer was observed in presence of fructose and glucose. In fact at the high additive

AC

concentrations, the monosaccharide additives have strong tendency to associate with themselves. The additive-additive interactions may discourage water structure breaking at the higher additive concentrations. It may be mentioned that the dye-additive hydrogen bond formation plays an important role in the spectral shift and fluorescence quenching of the dyes in solution.

25

ACCEPTED MANUSCRIPT

References: [1] F.J. Green, The Sigma-Aldrich Handbook of Stains, Dyes and Indicators, Aldrich, Milwaukee, 1991. [2] D. Sokic-Lazic and S.D. Minteer, Biosens. Bioelectron. 24 (2008) 945950.

IP

T

[3] L. Antonov, G. Gergov, V. Petrov, M. Kubista and J. Nygren, Talanta, 49 (1999)

CR

99106.

[4] S. Yamamoto, S. Kobashi, K. Tsutsui and Y. Sueishi, Spectrochim. Acta A. 66 (2007)

US

302–306.

[5] B. Boruah, P.M. Saikia and R.K. Dutta, Dyes Pigm. 85 (2010) 16–20.

AN

[6] F. Lopez Arbeloa, V. Martınez Martınez, T. Arbeloa, and I. Lopez Arbeloa, J.

M

Photochem. Photobio. C: Photochemistry Reviews, 8 (2007) 85–108.

ED

[7] M. Havelcová, P. Kubát and I. Němcová, Dyes Pigm. 44 (2000) 49–54. [8] A. Chakraborty, R.Adhikari, and S.K. Saha, J. Mol. Liq. 143 (2008) 81–88.

PT

[9] A. Ghanadzadeh, A. Zeini, A. Kashef and M. Moghadam, J. Mol. Liq. 138 (2008) 100–

CE

106.

[10] K. Patil, R. Pawar and P. Talap, Phys. Chem. Chem. Phys. 2 (2000) 4313–4317.

AC

[11] J. Vara and C.S. Ortiz, Spectrochim. Acta A. 166 (2016) 112–120. [12] S. Das and P.V. Kamat, J. Phys. Chem. B. 103 (1999) 209–215. [13] M.N. Usacheva, M.C. Teichert and M.A. Biel, J. Photochem. Photobiol. B. 71 (2003) 87 –98. [14] A. Chakraborty, M. Ali and S.K. Saha, Spectrochim. Acta A. 75 (2010) 1577–1583.

26

ACCEPTED MANUSCRIPT

[15] P.A. Bolotin, S.F. Baranovsky and M.P. Evstigneev, Spectrochim. Acta A. 64 (2006) 693 –697. [16] A. Cz'ımerová, J. Bujdák and A. Gáplovský, Colloids Surf. A Physicochem. Eng. Asp. 243 (2004) 89–96.

IP

T

[17] A. Ghanadzadeh, M.A. Zanjanchi and R. Tibandpay, J. Mol. Struct. 616 (2002) 167 –

CR

174. [18] S. Tunç, O. Duman, Fluid Phase Equilib. 251 (2007) 1–7.

US

[19] S. Tunç, O. Duman, B. Kancı, Dyes and Pigments, 94 (2012) 233-238. [20] O. Duman, S. Tunç, B. Kancı, Fluid Phase Equilibria, 301 (2011) 56–61.

AN

[21] M.C. Stumpe and H. Grubmuller, Biophys. J. 96 (2009) 3744–3752.

M

[22] F. Khan, D. Kumar, N. Azum, M.A. Rub and A.M. Asiri, J. Mol. Liq. 208 (2015) 84– 91.

ED

[23] U. Thapa and K. Ismail, J. Colloid Interface Sci. 406 (2013) 172 –177.

PT

[24] M.A. Kabir-ud-Din and A.Z. Rub, Naqvi, J. Surfactant Deterg. 15 (2012) 541 –550. [25] M. Ash, I. Ash, Industrial Chemical Additives, Synapse Information Resources, Inc.

CE

Electronic Handbook-2000 Edition.

AC

[26] J-S. Hsiung, Y-C. Huang, K-C. Li and S. Yang, J. Environ. Manage. 84 (2007) 384– 389.

[27] D.U. Lima, R.C. Oliveira and M.S. Buckeridge, Carbohydr. Polym. 52 (2003) 367– 373. [28] H.S. Frank and F. Franks, J. Chem. Phys. 48 (1968) 4746–4757. [29] T. Takahashi, H. Hoshino, and T. Yotsuyanagi, Anal Sci. 17(7) (2001) 847–851. [30] Y. Feng, Z-W. Yu, and P.J. Quinn, Chem. Phys. Lip. 114 (2002) 149–157.

27

ACCEPTED MANUSCRIPT

[31] F. H. Stillinger 209 (1980) Science 451–457. [32] Y. Marcus, Pure Appl. Chem., 82 (2010) 1889–1899. [33] Y. Marechal, The Hydrogen Bond and the Water Molecule,1st Edition, Oxford: Elsevier, 2007.

IP

T

[34] R.W. Gurney, Ionic Processes in Solution, McGraw–H ill, New York, 1953.

CR

[35] G.N.I. Clark, C.D. Cappa, J.D. Smith, R.J. Saykally, T. HeadGordon, 108 (2010) Mol. Phys. 1415–1433.

US

[36] J. L. England and G. Haran, Annu. Rev. Phys. Chem. 62 (2011) 257277.

AN

[37] M.C. Stumpe and H. Grubmüller. J. Am. Chem. Soc. 129 (2007) 1612616131. [38] H. Kokubo, J. Rösgen, D.W. Bolen, and B.M. Pettitt, Biophys. J. 93 (2007)

M

33923407.

ED

[39] C. Tanford, J. Am. Chem. Soc. 86 (1964) 2050-2059. [40] F. Ramondo, L. Bencivenni, R. Caminiti, A. Pieretti, and L. Gontrani, Phys. Chem.

PT

Chem. Phys. 9 (2007) 22062215.

179191.

CE

[41] L. Costantino, G. D`Errico, O. Ortona, and V. Vitagliano, J. Mol. Liq. 84 (2000)

AC

[42] B. Kallies, Phys. Chem. Chem. Phys. 4 (2002) 86–95. [43] R. Zangi, R. Zhou and B.J. Berne, J. Am. Chem. Soc. 131(4) (2009) 1535 –1541. [44] A. Caballero-Herrera, K. Nordstrand, K.D. Berndt, and L. Nilsson, Biophys. J. 89 (2005) 842–857. [45] A. Idrissi, Spectrochim. Acta A. 61 (2005) 1–17.

28

ACCEPTED MANUSCRIPT

[46] K.R. Lindfors, S.H. Opperman, M.E. Glover, and J.D. Seese, J. Phys. Chem. 76 (1971) 3313–3316. [47] A. Luttringhaus and H.W. Dirksen, Angew. Chem. Int. Ed. Engl. 3 (1964) 260–269. [48] G. Micheletti, C. Delpivo, and G. Baccolini, Green Chem. Lett. Rev. 6 (2013)

IP

T

135139.

CR

[49] L. Tavagnacco, O. Engström, U. Schnupf, M.-L. Saboungi, M. Himmel, G. Widmalm, A. Cesàro, and J.W. Brady, J. Phys. Chem. B. 116 (2012) 11701–11711.

US

[50] M.C. Gray, A.O. Converse, and C.E. Wyman, Appl. Biochem. Biotechnol. 105108

AN

(2003) 179193.

[51] T. Afrin, N.N. Mafy, M. M. Rahman, M. Yousuf, A. Mollah, and M.A.B H. Susan,

M

RSC Adv. 4 (2014) 50906–50913.

ED

[52] A. Ghanadzadeh Gilani, M. Moghadam, S.E. Hosseini, and M.S. Zakerhamidi, Spectrochim. Acta Part A. 83 (2011) 100–105.

PT

[53] A. Ghanadzadeh Gilani, T. Ghorbanpour, and M. Salmanpour, J. Mol, Liq. 177 (2013)

CE

273–282.

[54] A. Ghanadzadeh Gilani and S. Shokri, J. Mol, Liq. 193 (2014) 194–203.

AC

[55] A. Ghanadzadeh Gilani, H. Dezhampanah, and Z Poormohammadi-Ahandani, Spectrochim. Acta Part A. 179 (2017) 132–143. [56] C.S. Oliveira, K.P. Branco, M.S. Baptista, and G.L. Indig, Spectrochim. Acta Part A. 58 (2002) 2971–2982. [57] A. Ghanadzadeh Gilani, M. Moghadam, and M.S. Zakerhamidi, Comput. Methods Prog. Biomed. 104 (2011) 175 –181.

29

ACCEPTED MANUSCRIPT

[58] M. Kasha, H.R. Rawls, and A. El-Bayoumi, Pure Appl. Chem. 11 (1965) 371–392. [59] M. Kasha, Radiat. Res. 20 (1963) 55–71. [60] A. Eisfeld and J.S. Briggs, Chem. Phys. 324 (2006) 376 –384. [61] W.H. Brandeburgo, S.T van der Post, E.J. Meijer, and B. Ensing, Phys. Chem. Chem.

IP

T

Phys. 17 (2015) 2496824977.

CR

[62] C. Reichardt, Solvents and Solvent Effects in Organic Chemistry, third ed., Wiley VCH & Co, KGaA, 2003.

US

[63] R. Gupta and G. N. Patey, J. Chem. Phys. 141 (2014) 064502.

AC

CE

PT

ED

M

AN

[64] M.H. Abraham and R.W. Taft, J. Chem. Soc. Perkin Trans. 2 (1993) 305306.

30

ACCEPTED MANUSCRIPT

Table 1 The spectral data and the dimer association constants of azure B and toluidine blue in aqueous solution and in aqueous solutions of the molecular additive. J







U



cm

(°)

R (Å)

(D)

(D)

(D)

16802 16757 16689 16725 16694 16710

14560 14798 14859 14857 14868 14792

1121 980 915 934 913 959

44 49 48 47 46 45

6.1 6.2 7.0 6.9 7.1 6.9

7.87 7.63 8.51 9.09 9.23 9.14

34.28 44.48 45.75 45.42 44.29 48.39

3.30 4.73 4.09 5.39 5.31 5.78

0.4 0.4 0.4 0.4 0.4 0.4

15152 15175 15129 15152 15106 15015

16682 16697 16676 16641 16603 16598

14877 14781 14791 14806 14344 14801

903 958 943 918 1130 899

49 48 47 46 45 44

7.0 7.0 7.1 7.0 6.7 6.8

8.52 8.47 9.25 8.95 9.35 8.52

48.98 48.18 49.24 46.27 50.98 38.89

4.42 3.54 5.42 5.19 5.85 4.32

0.4 0.5 0.4 0.5 0.4 0.4

4064 3395 2253 1604 1339 1064

15221 15175 15198 15175 15175 15152

16778 16769 16658 16633 16626 16500

14746 14764 14743 14723 14791 14960

1.6 pure

1998 1556

15175 15221

16805 16840

0.01

1905

15221

16809

0.2 0.4 0.8 1.2 1.6

3548b 3041 2697 2080 1669 1437

15924 15873 15898 15848 15798 15848

17421 17451 17378 17334 17308 17392

14687 14571 14666 14684 14611 14462

1367 1440 1356 1325 1349 1465

43 43 42 41 40 39

6.0 6.3 6.2 6.5 6.7 6.6

0.2 0.4 0.8 1.2 1.6 3.0

2981 2515 1863 1480 1224 1000

15848 15848 15873 15823 15798 15723

17330 17336 17390 17487 17313 17246

14707 14754 14579 14170 14698 14651

1312 1291 1406 1659 1308 1298

43 42 41 40 39 38

0.2 0.4 0.8 1.2 1.6 3.0

2807 2345 1636 1224 973 765

15873 15898 15848 15873 15873 15823

17333 17317 17303 17291 17283 17215

14567 14622 14598 14536 14547 14581

1383 1348 1353 1378 1368 1317

Tetramethylurea

3.0 Pure

1588 1236

15924 15898

17487 17402

14631 14328

Acetylenediurea

0.01

1571

15924

17466

14671

Fructose

0.2 0.4 0.8 1.2 1.6 3.0

3429 2836 2253 1737 1510 1380

Urea

0.2 0.4 0.8 1.2 1.6 3.0

Tetramethylurea

Acetylenediurea

Urea

J

J /H

1016 1003 958 955 918 770

48 47 45 44 43 42

6.1 6.4 7.0 6.5 6.7 7.6

7.46 8.10 8.15 7.78 7.95 8.71

46.28 49.69 51.99 45.62 45.78 46.94

4.51 5.13 1.42 4.13 3.83 3.84

0.5 0.6 0.7 0.8 0.9 1.0

14633 14582

1086 1129

47 45

6.2 5.4

8.00 6.71

45.60 36.20

4.84 3.76

0.4 0.4

14675

1067

47

6.1

7.80

45.36

4.80

0.4

8.50 9.28 8.63 9.36 9.95 9.88

36.03 41.28 39.72 40.51 44.52 36.09

3.61 4.085 3.655 3.92 4.37 3.26

0.3 0.2 0.2 0.2 0.2 0.2

6.2 6.2 5.9 5.5 7.0 6.5

8.72 8.61 8.46 9.28 10.22 9.15

47.76 47.53 35.51 34.76 35.33 35.33

4.63 4.74 3.56 5.19 3.90 3.82

0.2 0.2 0.2 0.2 0.2 0.2

45 44 42 41 40 38

5.7 5.9 5.9 5.7 5.6 6.4

7.92 8.20 8.24 7.87 7.80 9.20

33.28 32.18 32.99 33.31 31.47 37.53

3.20 3.23 3.32 3.45 3.53 3.83

0.4 0.5 0.6 0.7 0.8 1.0

1428 1537

38 37

5.9 5.2

8.23 7.71

32.20 30.82

2.93 3.78

0.2 0.1

1398

37

6.1

8.67

34.63

3.04

0.2

M

ED

PT

CE

AC

Fructose

H

T

15152 15175 15175 15152 15152 15129

M

IP

4898a 4403 3572 2592 1998 1720

-1

CR

0.0 0.2 0.4 0.8 1.2 1.6

-1

US

cm

-1

AN

cm

Toluidine Blue (TB) water Glucose

b

H

( M 1 )

Azure B (AB) water Glucose

a

M

cm-1

Kd

C (M)

Media

1 4677 M [47]. 1 M 3311 [47].

31

ACCEPTED MANUSCRIPT

Table 2 Stokes shifts of azure B and toluidine blue in aqueous solution and aqueous solutions of the additives ([Additives] = 0.8 M) under ambient condition

Stokes shift ( cm 1 )

Dye

water + urea

water + fructose

water + glucose

Azure B

411

492

596

574

Toluidine Blue

656

970

944

IP CR

923

US AN M ED PT CE AC

32

T

water (pure)

TMU (pure) 449 680

ACCEPTED MANUSCRIPT

Figure captions: Fig. 1 Molecular structure of the (a) two sets of molecular additives and (b) phenothiazine dye. Fig. 2 Exciton energy diagram for monomer and dimer energy levels wit parallel, linear, and oblique electronic transition. Fig. 3 Variation of the absorbance of (a) azure B and (b) toluidine blue ( Cdye  5  105 M ) as a

IP

T

function of urea [U] concentration in water; (1) 0.4 M, (2) 0.8 M, (3) 1.2 M, (4) 1.6 M, (5) 3.0 M, (dashed spectrum is the absorbance of the dye in water).

US

CR

Fig. 4 Normalized absorption spectrum of (a) azure B and (b) toluidine blue in aqueous solution of the additives; urea (U), tetramethylurea (TMU), and thiourea (TU, the spectrum taken from Ref. 55), dashed spectrum is the absorbance of the dye in water; [Dye] = 1 104 M and [additive] = 1.6 M.

AN

Fig. 5 Variation of the absorbance of (a,b) azure B (AB) and (c,d) toluidine blue (TB) as a function of the concentration of aqueous solution of the monosaccharides (glucose and fructose [TU]; (15) 0.2 M, (2) 0.4 M, (3) 0.8 M, (4) 1.2 M, (5) 1.6 M, (dashed spectrum is the absorbance of the dye in water); [Dye] = 5  104 M

M

Fig. 6 Calculated monomer and dimer absorption spectra of (a,b) azure B (AB) and (c,d) toluidine blue (TB) in water and in the aqueous additive solutions.

ED

Fig. 7 Variation of (a) J- to H-band intensity ratio (  J /  H ) and (b-d) the excitonic parameters

PT

and the of azure B and toluidine blue as a function of urea (U) and thiourea (TU) concentrations in water; (the data for TU were taken from Ref. 55. Fig. 8 Effect of the additive concentration on the dimer association constant ( K d ) of (a) azure B

CE

and (b) toluidine blue a comparative additive effect. The K d values in aqueous solutions of thiourea were taken Ref. 55.

AC

Fig. 9 (a) Pure water structure, (b) dimerization of dye molecules in water, (c) additive-induced water structure and aggregate breaking at lower additive concentration, (d) additiveadditive association at higher additive concentration. Fig. 10 Molar absorptivities of (a) azure B and (b) toluidine blue in aqueous pure tetramethylurea [TMU]; [Dye] = (1) 1  105 M , (2) 3  105 M , (3) 5  105 M , (4) 1  104 M , (5) 2  104 M , (6), 3  104 M (7) 5  104 M . Fig. 11 Absorption and fluorescence spectra of (a) azure B and (b) toluidine blue in the aqueous solutions of additives ([additive] = 0.8 M); 1) pure water, 2) urea, 3) glucose, 4) fructose. Fig. 12 Fluorescence spectra of (a) azure B and (b) toluidine blue in the aqueous solution and aqueous solutions of urea [U]; (1) water, (2) [U] = 0.8 M, (3) [U] = 1.6 M.

33

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

M

AN

US

CR

IP

T

Fig. 13 Absorption and fluorescence spectra of (a) azure B and (b) toluidine blue in pure tetramethylurea at room temperature

34

ACCEPTED MANUSCRIPT

Fig. 1 O O H2N

C

H3C NH2

N

CH3

CH3

Tetramethylurea

Acetylenediurea

OH

OH

O

HO HO

H

OH

OH

D-glucose

OH

OH

US

OH

O

CR

OH

IP

T

Urea

CH3

N

D-(-) Fructose

N

H2N

S

N

1/2 ZnCl2

ED

H3C

M

AN

(a)

N

CH3

H N

CH3 Cl

N

Azure B

AC

CE

(b)

35

CH3

CH3 Cl

CH3

Toluidine Blue O

PT

S

ACCEPTED MANUSCRIPT

Fig. 2

E2

E2

E

E

E E1

G Monomer

Blue shift

Dimer

Band splitting

θ

Oblique-dimer (intermediate structure)

AC

CE

PT

ED

M

AN

H-dimer (parallel structure)

US

α

36

Monomer

CR

Dimer

IP

G

Monomer

E1

T

E1

G

E2

Dimer

Red shift

J-dimer (linear structure)

ACCEPTED MANUSCRIPT

Fig. 3

2

5

[AB] = 0.0005 M + U

4 3

1.5

2

T

Absorbance

1

IP

w

CR

1

US

0.5

0 450

550

650

750

AN

Wavelength (nm)

M

(a)

[TB] = 0.0005 M + U

5 4 3 2 1

w

PT

Absorbance

1.5

ED

2

CE

1

AC

0.5

0 450

550

650

Wavelength (nm)

37

750

ACCEPTED MANUSCRIPT

Fig. 4

1.1

[AB] = 0.0001 M

w TM U U TU

CR

IP

Absorbance

T

0.7

US

0.4

0.0 450

550

650

750

AN

Wavelength (nm)

M

(a)

1.1

w

ED

[TB] = 0.0001 M

U TU

CE

Absorbance

PT

0.7

TM U

AC

0.4

0.0 450

550

650

Wavelength (nm)

(b)

38

750

ACCEPTED MANUSCRIPT

Fig. 5

2

2.0 [AB] = 0.0005 M+ Glu

[AB] = 0.0005 M + Fru

1

0.5

0.5

0.0

450

550

650

750

Wavelength (nm)

450

AN

(a)

US

0

0

550

Absorbance

1

(b)

[TB] = 0.0005 M + Fru

1 w

1.0

0.5

0.0 650

750

450

Wavelength (nm)

AC

450

CE

0.5

750

1.5

PT

1

ED

w

650

Wavelength (nm)

5

5

1.5

550

2.0

M

[TB] = 0.0005 M + Glu

2

Absorbance

T

1.0

IP

1

w

CR

1

w

5

1.5

5

Absorbance

Absorbance

1.5

550

650

Wavelength (nm)

(c)

(d)

39

750

ACCEPTED MANUSCRIPT

Fig. 6 9 w

AB

9

Fru

AB

Dimer

ADU

-1

6

e / 10 ( M

3

CR

4

4

e / 10 ( M

3

J-band

0 550

650

US

0

450

750

450

Wavelength (nm)

6

CE 550

-1

. cm )

Monomer 6

Fru

H-band

3

J-band

J-band

0

650

750

450

Wavelength (nm)

AC

Glu

4

PT

e / 10 ( M

4

450

(b)

-1

U

0

750

Dimer

e / 10 ( M

ED

4

-1

-1

. cm )

w

2

650

Wavelength (nm)

TB

M

Monomer

H-band

550

9

ADU

Dimer

J-band

AN

(a)

TB

Monomer

H-band

-1

Monomer

T

U

H-band

IP

6

. cm )

Glu

-1

-1

. cm )

Dimer

550

650

Wavelength (nm)

(c)

(d)

40

750

ACCEPTED MANUSCRIPT

Fig. 7

1600

1.2 AB + T U

TB + U

TB + TU AB + U

1300

0.9

 J/ H 0.6

IP

-1

T

Ū (cm )

TB + U

0.3

CR

1000

1

2

US

700

0

3

0

[Additive] (M)

8

2

3

[U] (M)

(b)

AN

(a)

1

AB + U

49

M

AB + U

R (Å)

ED

7

45

 o

PT

TB + U

5 0

CE

6

1

41 TB + U

37 2

3

[U] (M)

AC

AB + U

0

1

2

[U] (M)

(c)

(d)

41

3

ACCEPTED MANUSCRIPT

Fig. 8 8.6 AB 8.2

7.8

T

IP

ln Kd

T MU Glu

7.4

CR

Fru

U

7.0

US

TU

6.6 0.0

1.0

2.0

3.0

AN

[Additive] M

(a)

TB

T MU

PT

ln Kd

7.8

ED

M

8.3

CE

7.3

Glu Fru

AC

6.8

U

TU

6.3 0.0

1.0

2.0

[Additive] M

(b)

42

3.0

ACCEPTED MANUSCRIPT

CR

IP

T

Fig. 9

(b)

M

AN

US

(a)

(d)

AC

CE

PT

ED

(c)

43

ACCEPTED MANUSCRIPT

Fig. 10 3.5 7

AB in pure TMU

6

3

4

2.5

T

1.5

2 1

IP

2

CR

-1

3

4

-1

e / 10 ( M . cm )

5

1

US

0.5 0 450

550

650

750

AN

Wavelength (nm)

(a)

M

3.5

TBO in pure TMU

1 2 3 4 5 6 7

1.5

CE

4

 / 10 ( M

PT

-1

2

ED

2.5

. cm

-1

)

3

1

AC

0.5 0 450

550

650

Wavelength (nm)

(b)

44

750

ACCEPTED MANUSCRIPT

Fig. 11

1.2

1.2 AB 2 3,4

0.8

CR

IP

0.8

0.4

0.4

520

560

600

AN

0

640

680

Fluorescence (a.u)

T

1

US

Absorbance (a.u)

1,2,3,4

0 720

Wavelength (nm)

M

(a)

32 4

0.8

0.8

0.4

AC

0.4

0

0 520

570

620

Wavelength(nm)

(b)

45

670

720

Fluorescence (a.u)

1

PT

1,2,3,4

1.2

CE

Absorbance (a.u)

TB

ED

1.2

ACCEPTED MANUSCRIPT

Fig. 12 60 AB + U 1

2

Flourescence

40

IP

T

3

US

CR

20

0 670

680

690

700

710

720

AN

Wavelength (nm)

240

ED

160

1

2

PT

Intensity (arb.)

TB + U

M

(a)

CE

3

AC

80

0 600

640

680

Wavelength (nm)

(b)

46

720

ACCEPTED MANUSCRIPT

Fig. 13 1.2 AB

IP

T

Intensity (a.u)

0.8

CR

0.4

Emission

US

Absorbance

0 520

570

620

670

720

AN

Wavelength (nm)

(a)

PT

Intensity (a.u)

0.8

ED

TB

M

1.2

AC

CE

0.4

Absorption

Emission

0 520

570

620

Wavelength(nm)

(b)

47

670

720

IP

T

ACCEPTED MANUSCRIPT

(b)

M

AN

US

CR

(a)

(d)

ED

(c)

AC

CE

PT

Graphical abstract

48

ACCEPTED MANUSCRIPT

Research Highlights Spectral behavior of phenothiazine dyes in aqueous solutions of ureas and monosaccharides was studied. Dimerization constants for the dyes were determined.

T

Structure of dimers in the aqueous additive media was interpreted.

IP

Effect of the additives on the competition of H-type and J-type dimers was studied.

AC

CE

PT

ED

M

AN

US

CR

The role of dye-additive interactions on the dye spectral characteristics was studied

49