1-s2.0-S0022286015004354-main

7232019 1-s20-S0022286015004354-main

httpslidepdfcomreaderfull1-s20-s0022286015004354-main 18

The self-aggregation of some 7H-indolo[12-a]quinolinium dyes in

aqueous solution

Marta J Sawicka

Department of Instrumental Analysis Faculty of Chemical Technology and Engineering West Pomeranian University of Technology Szczecin Piast ow Ave

42 71-065 Szczecin Poland

a r t i c l e i n f o

Article history

Received 28 November 2014

Received in revised form

13 May 2015

Accepted 13 May 2015

Available online 28 May 2015

Keywords

Merocyanine

Absorption spectra

Aggregate

Equilibrium constant

Ionic strength

a b s t r a c t

The aggregation of some 7H-indolo[12-a]quinolinium dyes in aqueous solution was investigated based

on UV evis spectroscopy The in1047298uence of pH dye concentration ionic strength and the addition of

organic solvents on the process was studied The aggregation took place in alkaline solution and was

strongly affected by ionic strength and the presence of organic solvents The existence of aggregates in

the solution was manifested in a new band appearance which exhibited bathochromic shift with respect

to the monomer band For all studied dyes the band was assigned to the dimer and the equilibrium

constants for dimer formation were calculated The dye tendency to aggregate was determined by its

structure and was enhanced by the presence of naphthalene moiety in comparison to that of benzene

ring as well as electron-rich substituents such as halogens

copy 2015 Elsevier BV All rights reserved

1 Introduction

Some groups of dyes such as cyanines merocyanines or some

pyrylium or thiopyrylium laser dyes tend to aggregate in concen-

trated or even in diluted solutions [1e3] Aggregation affects their

colouristic and photo-physical properties and is therefore of special

interest due to its possible application in understanding and

interpretation of a great variety of problems such as energy

transfer in biological systems conformation of polypeptides

staining properties of dyes for biological specimens Moreover the

phenomenon can be very useful for analytical application in optical

sensor adsorption and photography [4e6]

Many theories have been suggested to explain the forces of

attraction between ionic dyes in solution [78] The aggregation

have been attributed to van der Waals forces ion-dipole and

dipoleedipole interactions and forces arising from delocalized p-

electrons The mutual repulsion forces are reduced by the inclusion

of counter ions [9] According to previous studies the dyeedye

interactions are not the major driving force behind the aggregation

but strong waterewater interactions force the dye to aggregate

[10] The increase in the molecular size or the concentration of

solute leads to the destruction of the water network since the

number of water molecules is not big enough to form a complete

shell of hydration especially when the dipole character of a dimer

is smaller than that of a monomer In more concentrated solution

the destruction of the shell of hydration facilitates the dyeedye

contact and the same the aggregate creation [11]

The UV evis absorption spectroscopy is one of the most suitable

methods for studies of the aggregation process since the aggregates

in solution usually exhibit distinct changes in the absorption

spectrum in comparison to the monomeric species Quantitative

studies of aggregation properties of dyes are the most often con-

ducted in the concentration range 103e 106 mol L 1 in which

usually monomeredimer equilibrium exists The values of aggre-

gation constant determined for various dyes are of the order of

103e104 L mol1 [245812]

The aggregation behaviour of cyanine dyes has been studied

extensively since these are the best-known self-aggregating dyes

[1314] The structurally related dyes to cyanines are mer-

ocyanines These are systems in which an electron-donating

group D is linked by a conjugated system R to an electron-

accepting group A Their intermediate p-electronic structure of

a ground state can be described in terms of two mesomeric

structures DeR eA4 DthorneR eA- differing in a charge distribution

and bonds length and as a consequence in a dipole moment andE-mail address msawickazutedupl

Contents lists available at ScienceDirect

Journal of Molecular Structure

j o u r n a l h o m e p a g e h t t p w w w e l s e v ie r c o m l o c a t e m o l st r u c

httpdxdoiorg101016jmolstruc201505032

0022-2860copy

2015 Elsevier BV All rights reserved

Journal of Molecular Structure 1098 (2015) 26e33

7232019 1-s20-S0022286015004354-main


solvent shell The 1047297rst one is a quinoid form and the second e a

zwitterionic one (also named as betaine) [15] Among the mer-

ocyanines 7H-indolo[12-a]quinolinium dyes are worth

mentioning [16e22] They are usually generated in situ from

perchlorates of corresponding hydroxyaryl compounds via treat-

ing them with anhydrous potassium carbonate These mer-

ocyanines exhibit distinct solvatochromic properties which

means that their UV evis absorption spectra strongly depend on

the polarity of the medium Therefore they have found many

applications for instance in the determination of the ternary

solvent mixtures composition [2324] in investigations of the

diffusion pro1047297le in polymers [2526] as well as in quantitative

analysis of ionic surfactants [27e30] These investigations con-

cerned their monomeric forms

In this paper the aggregation behaviour of some 7H-indolo[12-

a]quinolinium merocyanines in alkaline aqueous solutions is

described for the 1047297rst time The dyes used in the study were 5-(4-

hydroxyphenyl)-77-dimethyl-7H-indolo[12-a]quinolinium

perchlorate (IQHH) 5-(3-bromo-4-hydroxyphenyl)-77-dimethyl-

7H-indolo[12-a]quinolinium perchlorate (IQBH) 5-(35-dibromo-

4-hydroxyphenyl)-77-dimethyl-7H-indolo[12-a]quinolinium

perchlorate (IQBB) 5-(6-hydroxynaphthyl-2)-77-dimethyl-7H-

indolo[12-a]quinolinium perchlorate (IQNH) and 5-(5-bromo-6-hydroxynaphthyl-2)-77-dimethyl-7H-indolo[12-a]quinolinium

perchlorate (IQNB) The structures of these dyes are depicted in

Scheme 1 Aggregation was studied by UV evis absorption spec-

troscopy The in1047298uence of dye concentration pH of the solution

ionic strength and the addition of an organic solvent on the for-

mation of aggregates was analysed

2 Materials and methods

21 Materials

The 7H-indolo[12-a]quinolinium dyes were synthesised ac-

cording to the method described in the literature [16e19] NaOH

and NaCl were purchased from ChemLand DMSO for spectroscopy

methanol and DMF analytical grade were purchased from POCh

(Gliwice) Distilled water was generated in glass Buumlchi apparatus

and was used without additional puri1047297cation

22 Spectrophotometric measurements

UV evis absorption spectra were recorded with computer-

controlled spectrophotometer Specord M40 (Carl Zeiss Jena Ger-

many) modi1047297ed by Medson Electronics Co Ltd in the

12000e27000 cm1 range at temperature 25 plusmn 01 C Gas-tight

quartz cells with 5 cm 1 cm and 01 cm path length were used

The concentration-dependence measurements dye solutions

(106$105e 212$104 mol L 1) were prepared by adding appro-

priate amount of the dye concentrate (15 mg of the dye in the form

of perchlorate and 075 mL of anhydrous DMSO) to the 002 2

and 8 ww NaOH solution (pH 115 135 and 139) respectively at

constant concentration of DMSO (074 vv) It was checked that

the in1047298uence of this amount of DMSO on the shape and the position

of absorption bands corresponding to monomer and aggregate

does not exceed the instrumental error

The measurements of the organic solvent effect to 2 mL of IQNB

solution (882$105 mol L 1) in 2 NaOH 25 50 100 150 225 and

300 mL of methanol or DMF were added After each step of the

organic solvent addition the absorption spectra were measured

Additionally the same amounts of water were added to the dye

solution Since the shape of the absorption band did notchange and

its position was shifted less than 200 cm1 it was concluded that

the dilution of the alkaline dye solution could not be the reason of

the observed spectral changes

The measurements of the ionic strength in 1047298uence the dye solution

(005 g L 1) in water containing 002 NaOH was mixed with the

dye solution (005 g L 1) in NaCl solution (362 mol L 1) containing

002 NaOH in the ratio 10 31 11 13 and 01

3 Results and discussion

31 The effect of the dye concentration

UV evis spectra of aqueous solution of IQNB were measured in

concentration range 106$105e 212$104 mol L 1 at four pH

values The sample spectra are shown in Fig 1aec At pH 55 the

dye exists in the solution as a salt (perchlorate) since this pH value

is below the pKa of the dye which equals to 727 [19] The band

corresponding to perchlorate is located at 24721 cm1 (spectra not

shown) In this case any spectral changes with increasing dye

concentration cannot be observed After pH exceeds the pKa value

of the dye dissociation of hydroxyl group occurs and the dye exists

as a merocyanine In UV e

vis spectrum the band corresponding tosalt disappears and the new band appears at 20904 cm1 At pH

115 the band attributed to merocyanine ful1047297ls the LamberteBeer

law up to concentration 317$105 mol L 1 Therefore one can

admit the dye exists as a monomer However the further increase

in the dye concentration leads to a slight bathochromic (red) shift

(Fig 1a)

At pH 135 the increase in the dye concentration causes a

distinct red shift (Fig 1b) It results from the decrease in the in-

tensity of the monomer band (the band at 20904 cm1) which is

accompanied by the rise in the intensity of the new low-energy

band (the band at 18408 cm1) Since the new band appears as

the concentration of the dye increases it can be attributed to the

aggregate If the solution is even more alkaline (at pH 139) the

Scheme 1 The structures of 7H-indolo[12-a]quinolinium dyes

MJ Sawicka Journal of Molecular Structure 1098 (2015) 26 e 33 27

7232019 1-s20-S0022286015004354-main


band in long-wave part of the spectrum is a dominant one which

proves that the concentration of the merocyanine in form of

monomer is really low even in diluted dye solution ( Fig 1c)

The further increase in either dye concentration or pH value leads

to the appearance of precipitate which can be noticed in UV evis

spectrum as the rise of the base line and decrease in the intensity of

the absorption band To check if the precipitate contains dye ag-

gregates only the concentrated dye solution (995$104 mol L 1) in

20 NaOH was centrifuged the solution was decanted and the

residue was gradually re-dissolved in water The recorded UV evis

absorption spectra of the solutions obtained do not demonstrate any

changes in comparison with the spectra described above

The electronic absorption spectra were measured of aqueous

solutions of IQNH IQBB IQBH and IQHH respectively at pH 115135 and 139 at increasing dye concentration (16$105e

2$104 mol L 1) The sample spectra at pH 115 and 139 are shown

in Fig 2aed It is evident that the spectral changes are not identical

in case of all studied dyes IQNH ful1047297ls the LamberteBeerlawin the

whole studied concentration range at pH 115 whereas the aggre-

gation is observed at pH 135 and 139 In the spectra of IQBB and

IQBH the low-energetic band corresponding to aggregate formation

appeared at pH 139 only In case of IQHH the changes are the least

evident at any pH values

32 The in 1047298uence of organic solvent

For studying the effect of organic solvents two solvents differing

in polarity ie methanol (E

N

T frac14 0762 [15]) and DMF (E

N

T frac14 0404

[15]) were added to IQNB aqueous solution at pH 135 and the

spectral changes were investigated

As it can be seen in Fig 3 the addition of the organic solvent

induces the hypsochromic (blue) shift of the absorption band

resulted from the rise of the intensity of the band attributed to

monomeric merocyanine Since it is known that organic solvents

hinder aggregation this experiment can con1047297rm that the observed

spectral changes result from the aggregationdisaggregation

phenomenon

33 Determination of equilibrium constant

The observed spectral changes ie the decrease in the intensity

of the monomer band accompanied with the rise in intensity of the

new band which are easy to noticed with increasing dye concen-

tration as well as the increase in intensity of the monomer band

after organic solvent addition are typical for aggregates It allows

the obtained results to be analysed in terms of equilibrium between

monomer and aggregate form of the dye

According to the reaction scheme

aM Aa (1)

where M Aa and a denote monomer aggregate and the number of

monomer units in aggregate respectively the equilibrium constant

K can be calculate as follows

K frac14 frac12 Aa

frac12M a (2)

Due to the fact that the concentrations of monomer [M ] and

aggregate [ Aa] are related with the total concentration of the dye

[D] according to the equation

frac12D frac14 frac12M thorn afrac12 Aa (3)

the concentration of the aggregate can be expressed as follows

frac12 Aa frac14 frac12D frac12M

a (4)

Introduction of the Equation (4) into the Equation (2) and its

rearrangement into the logarithm form leads to the linear functions

obtaining

log K frac14 logethfrac12D frac12M THORN log a a logfrac12M (5)

and

logethfrac12D frac12M THORN frac14 a logfrac12M thorn const (6)

From the slope of the plot described by the Equation (6) it is

possible to evaluate the a value which enable the equilibrium

constant to be calculated as wellThe concentrations of the monomer being in the equilibrium

with the aggregate at different total dye concentrations could not

be calculated from LamberteBeer law in case of all studied com-

pounds since the absorption bands attributed to the monomer and

the aggregate respectively are partially overlapped Assuming that

only two forms (the monomer and one type of aggregate) exist in

the solution the calculations of the concentration of particular

species can be done according to the equation

An

l frac14 frac12M $εM

n

thorn frac12 Aa$ε Aan

(7)

where An is the absorbance value at a given wavelength n εM

n

is the

molar extinction coef 1047297

cients of the monomer at the wavelength n

Fig 1 Absorption spectra of IQNB measured at pH 115 (a) 135 (b) and 139 (c)

respectively dye concentrations 176$105 mol L 1 (1) 882$105 mol L 1 (2)

882$104 mol L 1 (3) path length 5 cm (1) 1 cm (2) 01 cm (3)

MJ Sawicka Journal of Molecular Structure 1098 (2015) 26 e 3328

7232019 1-s20-S0022286015004354-main


and ε Aan

is the molar extinction coef 1047297cients of the aggregate at the

wavelength n and l is the thickness of analyzed layer (path length)

εM n

values for each studied dye are available from the spectra

measured at pH 115 for diluted dye solution (the dyes concen-

tration amounts to 2$105 mol L 1) In this case only monomeric

form of the merocyanine is present in the solution and its con-

centration [M ] is equal to the total dye concentration [D] On the

other hand in concentrated dye solution at pH 135 the aggregate is

assumed to be the only form of IQNB IQNH IQBB and IQBH mer-

ocyanines ([M ] frac14 0) However the ε Aan

cannot be calculated from

the UV-VIS spectra due to both the a value and as a consequence

[ Aa] are unknown Therefore an apparent molar extinction co-

ef 1047297cients of the aggregateε Aan

was introduced which was calculated

from the spectra recorded at pH 139 and [D] equals to

2$104 mol L 1 as follows

ε

Aan

frac14 A

n

frac12D (8)

and the Equation (7) was rearranged into the form

An


n

thorn X $ε

Aan

(9)

where X $ε Aan

frac14 frac12 Aa$ε Aan

X frac14 a$frac12 Aa and ε

Aan

frac14 ε Aan

=a

According to Equation (9) the concentration of the monomer

[M ] at different total dye concentrations [D] were determined bymultiple regression analysis The calculations were done based on

the spectra recorded at pH 115 for IQNB at pH 135 for IQNB IQNH

and at pH 139 for IQNB IQNH IQBB IQBH The concentrations of

the monomer were determined with high accuracy The values of

correlation coef 1047297cients exceed 0999 (or more) and the values of

standard deviations calculated in absorbance unit were 35$103 (or

less) The evaluated values of [M ] were applied into the Equation (6)

and plots describing log ([D] [M ]) as a function of log [M ] were

created The results obtained at pH 139 are presented in Fig 4

The obtained plots for each case follow the straight line with

the correlation coef 1047297cient r at least 097 and the slope amounts to

20 plusmn 01 Therefore one can admit that the band in the long-wave

part of the UV-VIS spectrum of all studied dyes can be assigned to

Fig 2 UV-vis spectra of IQNH (a) IQBB (b) IQBH (c) and IQHH (d) measured at pH 115 (dotted line) and 139 (solid line) respectively dye concentrations (mol L 1) 2$105 (1)

5$105 (2) 8$105 (3) 12$104 (4) path length 1 cm

Fig 3 UV evis spectra of IQNB (882$105 mol L 1) measured in 2 NaOH e methanol

(a) and 2 NaOH e DMF (b) mixture organic solvent concentration ( vv) 0 (1) 123

(2) 244 (3) 476 (4) 698 (5) 1011 (6) 1304 (7)

Fig 4 Determination of the number of monomer units in aggregate a at pH 139 for

IQNB (rhombuses) IQNH (squares) IQBB (triangles) IQBH (cycles)


7232019 1-s20-S0022286015004354-main


the dimer The equilibrium constants for dimer formation (the

dimerisation constant) which were calculated from the equation

K frac14frac12Dfrac12M

2

frac12M 2 (10)

are collected in Table 1

34 The ionic strength in 1047298uence

The aggregate formation was found in aqueous solutions of

studied dyes at high pH value especially at pH 135e139 So small

differences in the pH were induced by signi1047297cant increase in NaOH

concentration (from 2 to 8 ww) which leads to the increase in

ionic strength of the solution as well To establish whether OH

anions are required to aggregate formation or the aggregation is

induced by the increase in the ionic strength of the solution elec-

tronic absorption spectra were measured of IQNB and IQBB solu-

tions respectively as a function of NaCl concentration at pH 115

(Fig 5)

In both cases the new band appeared the intensity of which

rose with increasing salt concentration It proves that the ionic

strength induces the dye aggregation making the water lattice

more rigid Moreover this tendency as well as the rise of the base

line in UV evis spectrum additionally con1047297rm the fact that the

observed spectral changes are caused by the creation of

aggregates

It is well known that water molecules create ordered structures

The structure of liquid water has been intensively investigated and

most models of water can be partitioned into two broad categories

(a) mixture models and (b) distorted hydrogen bond or ldquocontinuum

modelsrdquo [3132] The mixture models postulate the simultaneous

existence of two distinct types of structures In the continuum

models liquid water comprises a random three-dimensional

network of hydrogen bonds encompassing a broad distribution of

OeHO hydrogen bond (HB) angles and distances but the water

networks cannot be ldquobrokenrdquo or separated into distinct molecular

species as in the mixture models Many studies proved that in bi-

nary water e organic solvent mixture in water-rich region the

water molecules tend to prefer the interactions between them-

selves via hydrogen bond than with molecules of other species [33]

The small contaminants are closed in a sphere consists of about 24

molecules of water [34] The studies of Marcus [3536] had

demonstrated that some co-solvents does enhance the internal

water structure in water-rich region of the mixture whereas other

many of which are strongly hydrogen bonded with water does not

Chandra [37] found a reduction in the number of H-bonds with

increasing concentration of ions (NaCl and KCl) indicating that

water molecules are signi1047297cantly in1047298uenced by the presence of

ions Suresh with co-workers [38] concluded that it can result from

the steric hindrance of the ion or due to the high dipole ordering bythe ionic 1047297eld

In case of the studied merocyanines at low ionic strength the

monomer is the only form of the dye or the strongly predominant

one It indicatesthat the solvation by watertakes place of small ions

as well as of the dye molecules Since the merocyanine competes

against other ions for water molecules the increase in NaOH or

NaCl concentration leads to the reduction of dyeewater in-

teractions As a consequence in the solution of high ionic strength

the dye molecule tends to interact with other dye molecule rather

than with water thus the aggregates appear

Therefore the pH value as high as 135e139 seems to be not

necessary to aggregate formation but the high concentration of

ions favours the aggregation The formation of aggregate is possible

at pH higher than pKa of the dye which enable the whole popula-

tion of the dye exists in the form of zwitterionic merocyanine The

lack of zwitterionic form in case of hydroxyaryl perchlorate hinders

the formation of the stable aggregates It is well known that pH

value strongly in1047298uences the aggregation tendency of other dyes

especially if they have a substituent such as amine or hydroxyl

group which can be protonated or undergo dissociation at different

pH values [39e41] The tendency to aggregate of merocyanine (at

pH higher than pKa) and the lack of it in case of hydroxyaryl

perchlorate (at pH lower than pKa) suggests that electrostatic in-

teractions play an important role in aggregate formation These

interactions concern the ionized hydroxyl group and the indolo-

quinolinium moiety with positive charge at nitrogen atom For thatreason it can be assumed that merocyanine molecules in aggregate

are antiparallel stacked which enables the part of the merocyanine

molecule with the positive charge to face the part of the molecule

with the opposite one

The changes in UV evis absorption spectra which are observed

in the solution of aggregating dyes are the most often interpreted

based on the Kasha exciton theory [4243] According to this theory

two type of aggregates are distinguished face-to-face H -aggregates

with the absorption band blue shifted in comparison to the band

attributed to monomer and face-to-tail J -aggregates with the red

shifted aggregate band It is agreed that both H- and J-aggregates

are composed of parallel dye molecules but they differ one from

another in the slip angles ie angle between the line of centres of a

column of dye molecules and the long axis of any one of the parallelmolecules A small slip angle is attributed to the J aggregates and a

large slip angle e to the H aggregates [1344] Merocyanine dyes

preferentially form face-to-face-stacked centrosymmetric H-type

dimers [4445] It is possible due to almost planar structure of a dye

in which a donor (acceptor) part of one molecule is located directly

under an acceptor (donor) part of the other molecule Nevertheless

examples of J-aggregating merocyanines have been also described

especially in LB 1047297lms [46] as well as in the solution [4447e50] with

a slip of one molecule against each other along the direction of the

long molecular axis

Since in case of the studied dyes red shift of absorption band

during aggregation is observed the antiparallel stacking of two

molecules in dimer seems to be quite probable

Besides H and J aggregates theory the observed spectral shiftscan be related to the structure of a dimer taking into account the

changes of the molecule dipole moment occurring during aggre-

gation as well as the soluteesolvent interactions Both factors

Table 1

Aggregation characteristic of studied dyes at different pH value

pH 115 135 139

dye IQNB IQNB IQNH IQNB IQNH IQBB IQBH

a 204 197 194 191 203 192 190

r 0979 0996 0998 0979 0970 0995 0999

na 6 7 7 6 6 8 7

K (L mol1) 947 3836 932 167$105 122$105 225$104 147$104

a

The number of experimental points


7232019 1-s20-S0022286015004354-main


determine the excitation energy and are especially important in

case of the dyes which exhibit solvatochromic properties

The solvatochromic dyes are compounds with electric dipole

moment in a ground state distinctly different from that in an

excited state In that case the polarity of the medium affects the

position and the shape of UV-VIS absorption bands The extent and

the direction of these changes with increasing solvent polarity are

determined by the dipole moments of the ground and excited

states The more polar excited state provides stronger interactionwith polar solvent which leads to the bathochromic shift (positive

solvatochromism) And analogously when a polar ground state is

stronger stabilised by the polar medium the hypsochromic shift of

the absorption band is observed (negative solvatochromism) [15]

All of the studied merocyanines exhibit negative sol-

vatochromism in polar solvents as well as in solvents of medium

polarity [182022] It means that they dipole moment in a ground

state is greater than that in an excited state The interactions be-

tween the monomeric polar zwitterionic merocyanine and a polar

solvent (such as water) diminish the energy of the ground state

which results in the increase in the excitation energy (Scheme 2)

During the dimerization the antiparallel stacking of the molecules

leads to the compensation of their dipole moments Therefore a

dimer form is much less polar than monomer which causes weaker

interaction with polar solvent As a consequence the transition

energy diminishes and the absorption band attributed to the dimer

is red shifted in relation to the one corresponding to monomer

Furthermore the decrease in the dipole moment of the mole-

cules during aggregation seems to be also the reason why organic

solvents hinder aggregation As it can be seen in Fig 3 the addition

of DMFresults in the spectral changes which are more evident than

these caused by more polar methanol According to Yazdani et al

[4] the dominant role of water as the most favourable solvent to

aggregation of ionic dyes is associated with its high dielectric

constant which reduces the repulsive forces between dye mole-

cules in the aggregate Thus the dye molecules prefer to interact

with themselves rather than with the molecules of water On the

other hand in case of studied dyes the charge compensation of

monomers which occurs during aggregation allows the aggregates

to be solvated by less polar solvent It makes the interactions be-

tween monomers weaker As a consequence aggregates break upand the monomeric molecules appear in the solution again It im-

plies that hydrophobic forces play an important role in aggregation

of the studied dye as well

35 The effect of the dye structure

The dyes used in these studies can be arranged in terms of their

tendency to aggregate formation as follows IQNB gt IQNH gt

IQBB gt IQBH gt IQHH Indeed in the same series one can roughly

ordered the expected hydrophobic character of the dyes but some

other factors have to be taken into account as well One can admit

that the dye ability to aggregate depends on the dye structure as

well as the type of substituents During the aggregation the

substituted or unsubstituted phenyl ring or naphthalene moietyfaces the indoloquinolinium part of the dye The factors which

facilitate the interactions between these two parts of molecule

such as pep interactions will enhance the aggregation ability

Therefore the presence of naphthalene moiety favours aggregation

due to greater area of pep interactions in comparison with the dyes

with phenyl ring This is the reason why the dyes with naphthalene

moiety exhibit the greatest tendency to aggregate

The comparison of the spectra of IQNB and IQNH as well as IQBB

and IQBH or IQHH indicates that the aggregation is enhanced also

by the electron-rich substituents such as halogens Apart from the

fact that halogens increases the hydrophobic part of the monomer

molecule they can interact with p electrons of indoloquinolinium

moiety via halogen bond providing the stabilization of the forming

aggregate The importance of the Ce

Xe

p interactions in crystalstructures was noticed by Bishop et al who described the X-ray

structures of many inclusion compounds [51e54] According to

studies of Mafud and co-workers [55] the CeXep interactions are

one of three types of interactions which enhance crystal packing of

trans-12-dibromo-2-styrylpyridine Moreover Swierczynski and

co-workers found a large number of fragments involved in non-

covalent interactions of aryl components with halogens in the

Cambridge Structural Database (CSD) [56] Theoretical and exper-

imental analyses have shown that the electron density around a

bonded halogen atom is not spherically but anisotropically

distributed negative charge is concentrated in the equatorial area

(ie perpendicular to the CeX axis) and positive charge along the

CeX bond which is called s-hole This effect known as polar

1047298attening is strongly enhanced by the presence of electron

Fig 5 UV evis spectra of IQNB (a) and IQBB (b) (005 g L 1) in NaCl solutions NaCl concentration (mol L 1) 000 (1) 090 (2) 181 (3) 271 (4) 362 (5)

Scheme 2 Changes in excitation energy versus solvent polarity for monomer (DEM)

and dimer (DEA2) E0i

is the ground state energy of monomer (M) and dimer (A 2) and

E1i is the excited state energy of monomer (M) and dimer (A 2) respectively


7232019 1-s20-S0022286015004354-main


withdrawing groups and also increasing in the order Cl lt Br lt I

[5758] Therefore it can be expected that the halogen bond is

especially strongin case of IQBB which in fact forms the aggregates

the most readily in comparison to the rest of merocyanines with

phenyl ring The presence of phenyl ring without any additional

substituents can be the reason that IQHH exhibits the lowest ag-

gregation tendency

Based on the assumption that monomer molecules are anti-

parallel stacked in aggregate there are a few possible structures of

the dimer Some of them are presented in Scheme 3 One of the

factors which in1047298uence the number of the possible structures is the

mutual arrangement of the indoloquinolinium parts of two

monomer molecules in dimer As a result various structures exist

which can be named ldquosynrdquo and ldquoantirdquo dimers differing in p e

peoverlapping and the distance between ionized hydroxyl group

with negative charge and the nitrogen atom with positive one

Moreover due to the possibility of the free rotation around the

single bond between indoloquinolinium part and benzene or

naphthalene moiety s-cis and s-trans rotamers can be considered as

well In the solution the probability of the existence of each of

these structures seems to be similar However taking into account

electrostatic interactions p e peoverlapping and CeXep in-

teractions which in1047298uences the stability of forming aggregates onecan assumed that the ldquoantirdquo dimer composed of two s-trans

monomers is the most probable one An attempt to con1047297rm if this

dimer structure really dominates in the solution failed The un-

successful separation of a single crystal of the dimer made impos-

sible the application of X-ray crystallography which is usually used

to the structure determination

4 Conclusion

The aggregation of some 7H-indolo[12-a]quinolinium mer-

ocyanines is reported It was found that the phenomenon is

strongly affected by ionic strength and the presence of organic

solvents Electrostatic interactions as well as hydrophobic forcesplay an important role in aggregate formation Merocyanine is the

only form of the studied dyes which can create the aggregates

especially at high ionic strength The dye tendency to aggregate is

determined by its structure p e p interactions and CeXep in-

teractions provide a stabilization of the forming aggregates

References

[1] T Taguchi S Hirayama M Okamoto New spectroscopic evidence for mo-lecular aggregates of rhodamine 6G in aqueous solution at high pressureChem Phys Lett 231 (1994) 561e568

[2] L Antonov G Gergov V Petrov M Kubista J Nygren UV e Vis spectroscopicand chemometric study on the aggregation of ionic dyes in water Talanta 49(1999) 99e106

[3] T Kotowski W Skubiszak JA Soroka KB Soroka T Stacewicz Pyrylium andthiopyrylium high ef 1047297ciency laser dyes J Lumin 50 (1991) 39e45

[4] O Yazdani M Irandoust JB Ghasemi Sh Hooshmand Thermodynamic studyof the dimerization equilibrium of methylene blue methylene green andthiazole orange at various surfactant concentrations and different ionicstrengths and in mixed solvents by spectral titration and chemometric anal-ysis Dyes Pigm 92 (2012) 1031e1041

[5] S De S Das A Girigoswami Environmental effects on the aggregation of some xanthene dyes used in lasers Spectrochim Acta A 61 (2005)1821e1833

[6] M Dakiky I Nemcova Aggregation of oorsquo-dihydroxyazo dyese1 Concen-tration temperature and solvent effect Dyes Pigm 40 (1999) 141e150

[7] RH Peters Textile Chemistry in The Physical Chemistry of Dyeing vol IIIElsevier Amsterdam 1975

[8] G Alberghina R Bianchini M Fichera S Fisichella Dimerization of CibacronBlue F3GA and other dyes in1047298uence of salts and temperature Dyes Pigm 46(2000) 129e137

[9] R Ambrosetti G Belluci R Bianchini Direct numerical approach to complexreaction kinetics the addition of bromine to cyclohexene in the presence of pyridine J Phys Chem 90 (1986) 6261e6266

[10] C Tanford The Hydrophobic Effect Wiley Interscience New York 1980 [11] LR Pratt D Chandler Theory of the hydrophobic effect J Chem Phys 67

(1977) 3683e3704[12] R Sabate J Estelrich Determination of the dimerization constant of pina-

cyanol role of the thermochromic effect Spectrochim Acta A 70 (2008)471e476

[13] GB Behera PK Behera BK Mishra Cyanine dyes self aggregation andbehaviour in surfactants A review J Surf Sci Technol 23 (1e2) (2007) 1e31

[14] H Tojo K Horiike K Shiga Y Nishina H Watari T Yamano Self-associationmode of a 1047298avoenzyme D-amino acid oxidase from hog kidney J Biol Chem260 (23) (1985) 12607e12614

[15] Ch Reichardt Solvents and Solvent Effects in Organic Chemistry third edWiley-VCH Weinheim 2003

[16] KB Soroka JA Soroka Photochemical synthesis of 7H-indolo[12-a]quinoli-nium salts e a new ring system Tetrahedron Lett 21 (1980) 4631e4632

[17] KB Soroka JA Soroka Photochemistry of hemicyanines Part III Synthesis of 5-(hydroxyaryl)-77-dimethyl-7H-indolo[12-a]quinolinium perchlorates anddetermination of their acidities Chem Scr 29 (1989) 167e171

[18] MJ Sawicka JA Soroka EK Wroblewska IK Zawadzka Synthesis and UV-

VIS study of a new strongly solvatochromic merocyanine-like dye withmodi1047297ed donor part Pol J Chem 80 (2006) 1337e1351

[19] MJ Sawicka JA Soroka M Gasiorowska A new method for the preparationof solvatochromic 5-(5-X-6-hydroxynaphthyl-2)-7H-indolo[12-a]quinoli-nium merocyanines Pol J Chem Technol 12 (2010) 17e22

[20] JA Soroka KB Soroka Solvatochromism of dyes Part I Solvatochromism of merocyanines Derivatives of the 7H-indolo[12-a]quinolinium system A newmodel of solvatochromism J Phys Org Chem 4 (1991) 592e604

[21] KB Soroka JA Soroka Solvatochromism of dyes Part III Solvatochromism of merocyanines in some binary mixtures of solvents SAeSABeSB a new modelof solvatochromism J Phys Org Chem 10 (1997) 647e661

[22] MJ Sawicka JA Soroka M Gasiorowska EK Wroblewska The spectroscopicbehavior of two new 5-(5-R-6-hydroxynaphthyl-2)-77-dimethyl-7H-indolo[12-a]quinolinium merocyanines in various solvents J Sol Chem 41 (2012)25e35

[23] MJ Sawicka JA Soroka Application of the calibration surfaces method inquantitative analysis of water e ethanol e methanol mixture Cent Eur JChem 11 (2013) 1239e1247

[24] JA Soroka KB Soroka Calibration surfaces in analysis of ternary mixturesChem Anal (Warsaw) 47 (2002) 95e112

[25] JA Soroka Z Rosłaniec EK Wroblewska Barwniki solwatochromowe wbadaniach struktury polimerow Cz I Pro1047297le dyfuzji w poli(tereftalaniebutylenu) Polimery 47 (2002) 828e832

[26] EK Wroblewska JA Soroka Z Rosłaniec Barwniki solwatochromowe wbadaniach struktury polimerow Cz II Pro1047297le dyfuzji w wybranych kopo-li(estroeterach) Polimery 50 (2005) 286e290

[27] EK Wroblewska M Gasiorowska JA Soroka 5-(3-Bromo-4-hydroxy-5-methoxyphenyl)-77-dimethyl-7H-indolo[12-a]quinolinium perchlorate as anew indicator for anionic surface active agents determination Tenside SurfDet 47 (2010) 119e122

[28] EK Wroblewska M Gasiorowska JA Soroka 5-(3-Bromo-4-hydroxy-5-methoxyphenyl)-77-dimethyl-7H-indolo[12-a]quinolinium perchlorate as anew indicator for anionic surface active agents determination Part II In1047298u-ence of pH on the titration results Tenside Surf Det 48 (2011) 127e129

[29] M Gasiorowska EK Wroblewska Two-phase titration method for cationicsurface active agents determination with use of 5-(3-bromo-4-hydroxy-5-methoxyphenyl)-77-dimethyl-7H-indolo[12-a]quinolinium perchlorate dye

Scheme 3 Schematic representation of the possible structures of dimer


7232019 1-s20-S0022286015004354-main


Tenside Surf Det 49 (2012) 23e25[30] M Gasiorowska EK Wroblewska Manual direct two-phase titration method

for anionic surface active agents determination with use of solvatochromicdye The modi1047297cation towards toxicity abatement Tenside Surf Det 49(2012) 97e99

[31] HE Stanley J Teixeira Interpretation of the unusual behavior of H2O and D2Oat low temperatures tests of a percolation model J Chem Phys 73 (1980)3404e3422

[32] Q Sun Raman spectroscopic study of the effects of dissolved NaCl on waterstructure Vib Spectrosc 62 (2012) 110e114

[33] DC Da Silva I Ricken MA do R Silva VG Machado Solute-solvent in-teractions in the preferential solvation of Brookers merocyanine in binarysolvent mixtures J Phys Org Chem 15 (2002) 420e427

[34] BBJ Linde JA Soroka M Borkowski Ultrasonic investigation of ldquopseudo-stablerdquo structure in water mixed Fortschritte Akust (2002) 708e709

[35] Y Marcus Water structure enhancement in water-rich binary solvent mix-tures J Mol Liq 158 (2011) 23e26

[36] Y Marcus Water structure enhancement in water-rich binary solvent mix-tures Part II The excess partial molar heat capacity of the water J Mol Liq166 (2012) 62e66

[37] A Chandra Effects of ion atmosphere on hydrogen-bond dynamics in aqueouselectrolyte solutions Phys Rev Lett 85 (2000) 768e771

[38] SJ Suresh K Kapoo S Talwar A Rastogi Internal structure of water aroundcations J Mol Liq 174 (2012) 135e142

[39] T Kolev BB Koleva S Stoyanov M Spiteller I Petkov The aggregation of themerocyanine dyes depending of the type of the counterions SpectrochimActa A 70 (2008) 1087e1096

[40] VV Serra SM Andrade MGPMS Neves JAS Cavaleiro SMB Costa J-aggregate formation in bis-(4-carboxyphenyl)porphyrins in water pH andcounterion dependence New J Chem 34 (2010) 2757

e2765

[41] A Navarro F Sanz Dye aggregation in solution study of CI direct red I DyesPigm 40 (1999) 131e139

[42] EG McRae M Kasha Enhancement of phosphorescence ability upon aggre-gation of dye molecules J Chem Phys 28 (1958) 721e722

[43] M Kasha HR Rawls MA El-Bayoumi The exciton model in molecularspectroscopy Pure Appl Chem 1 (1965) 371e392

[44] F Wuumlrthner TE Kaiser CR Saha-Meurooller J-Aggregates from serendipitousdiscovery to supra-molecular engineering of functional dye materials AngewChem Int 50 (2011) 3376e3410

[45] Z Chen A Lohr CR Saha-Moller F Wuumlrthner Self-assembled p-stacks of

functional dyes in solution structural and thermodynamic features ChemSoc Rev 38 (2009) 564e584

[46] S-i Kuroda J-aggregation and its characterization in Langmuir-Blodgett 1047297lmsof merocyanine dyes Adv Colloid Interface Sci 111 (2004) 181e209

[47] K Goto R Omae M Yamaji T Shinmyozu J-type aggregation of a simplemerocyanine skeleton spectral features and structure of 4-amino-6-oxopyrimidine J Photochem Photobiol A Chem 194 (2008) 92e96

[48] F Mizutani S-i Iijima K Tsuda The aggregation of merocyanine dye in so-lution and 1047297lm Bull Chem Soc Jpn 55 (1982) 1295e1299

[49] Y Kalisky D Williams J laser photolysis studies of spiropyran-merocyanine

aggregate formation in solution Chem Phys Lett 86 (1) (1982) 100e

104[50] S Yagai H Higashi T Karatsu A Kitamura Dye-assisted structural modula-

tion of hydrogen-bonded binary supramolecular polymers Chem Mater 17(2005) 4392e4398

[51] ANMM Rahman R Bishop DC Craig ML Scudder Piehalogen dimer in-teractions and the inclusion chemistry of a new tetrahalo aryl host OrgBiomol Chem 2 (2004) 175e182

[52] ANMM Rahman R Bishop DC Craig ML Scudder Pi-halogen dimers andV-shaped tetrahalo aryl inclusion hosts Cryst Eng Commun 4 (2002)510e513

[53] ANMM Rahman R Bishop DC Craig ML Scudder Analysis of piehalogendimer interactions present in a family of staircase inclusion compounds CrystEng Commun 5 (2003) 422e428

[54] R Bishop ML Scudder DC Craig ANMM Rahman SF Alshahateet The pie halogen dimer (PHD) interaction a versatile new construction unit forcrystal engineering Mol Cryst Liq Cryst 440 (2005) 173e186

[55] AC Mafud MT do Prado Gambardella AC Favero Caire Towards to thetrans-bromination of 2-styrylpyridine with a palladacycle intermediary andstructure analysis for trans-12-dibromo-2-styrylpyridine J Mol Struct 988(2011) 87

e90

[56] D Swierczynski R Luboradzki G Dolgonos J Lipkowski HJ Schneider Non-covalent interactions of organic halogen compounds with aromatic systems eanalyses of crystal structure data Eur J Org Chem 2005 (2005) 1172e1177

[57] H Cicak M ETH akovic Z Mihalic G Pavlovic L Racane V Tralic-KulenovicHydrogen and halogen bonding patterns and pep aromatic interactions of some 67-disubstituted 13-benzothiazoles studied by X-ray diffraction andDFT calculations J Mol Struct 975 (2010) 115e127

[58] M Fourmeigue Halogen bonding recent advances Curr Opin Solid StateMater Sci 13 (2009) 36e45


7232019 1-s20-S0022286015004354-main


solvent shell The 1047297rst one is a quinoid form and the second e a

zwitterionic one (also named as betaine) [15] Among the mer-

ocyanines 7H-indolo[12-a]quinolinium dyes are worth

mentioning [16e22] They are usually generated in situ from

perchlorates of corresponding hydroxyaryl compounds via treat-

ing them with anhydrous potassium carbonate These mer-

ocyanines exhibit distinct solvatochromic properties which

means that their UV evis absorption spectra strongly depend on

the polarity of the medium Therefore they have found many

applications for instance in the determination of the ternary

solvent mixtures composition [2324] in investigations of the

diffusion pro1047297le in polymers [2526] as well as in quantitative

analysis of ionic surfactants [27e30] These investigations con-

cerned their monomeric forms

In this paper the aggregation behaviour of some 7H-indolo[12-

a]quinolinium merocyanines in alkaline aqueous solutions is

described for the 1047297rst time The dyes used in the study were 5-(4-

hydroxyphenyl)-77-dimethyl-7H-indolo[12-a]quinolinium

perchlorate (IQHH) 5-(3-bromo-4-hydroxyphenyl)-77-dimethyl-

7H-indolo[12-a]quinolinium perchlorate (IQBH) 5-(35-dibromo-

4-hydroxyphenyl)-77-dimethyl-7H-indolo[12-a]quinolinium

perchlorate (IQBB) 5-(6-hydroxynaphthyl-2)-77-dimethyl-7H-

indolo[12-a]quinolinium perchlorate (IQNH) and 5-(5-bromo-6-hydroxynaphthyl-2)-77-dimethyl-7H-indolo[12-a]quinolinium

perchlorate (IQNB) The structures of these dyes are depicted in

Scheme 1 Aggregation was studied by UV evis absorption spec-

troscopy The in1047298uence of dye concentration pH of the solution

ionic strength and the addition of an organic solvent on the for-

mation of aggregates was analysed

2 Materials and methods

21 Materials

The 7H-indolo[12-a]quinolinium dyes were synthesised ac-

cording to the method described in the literature [16e19] NaOH

and NaCl were purchased from ChemLand DMSO for spectroscopy

methanol and DMF analytical grade were purchased from POCh

(Gliwice) Distilled water was generated in glass Buumlchi apparatus

and was used without additional puri1047297cation

22 Spectrophotometric measurements

UV evis absorption spectra were recorded with computer-

controlled spectrophotometer Specord M40 (Carl Zeiss Jena Ger-

many) modi1047297ed by Medson Electronics Co Ltd in the

12000e27000 cm1 range at temperature 25 plusmn 01 C Gas-tight

quartz cells with 5 cm 1 cm and 01 cm path length were used

The concentration-dependence measurements dye solutions

(106$105e 212$104 mol L 1) were prepared by adding appro-

priate amount of the dye concentrate (15 mg of the dye in the form

of perchlorate and 075 mL of anhydrous DMSO) to the 002 2

and 8 ww NaOH solution (pH 115 135 and 139) respectively at

constant concentration of DMSO (074 vv) It was checked that

the in1047298uence of this amount of DMSO on the shape and the position

of absorption bands corresponding to monomer and aggregate

does not exceed the instrumental error

The measurements of the organic solvent effect to 2 mL of IQNB

solution (882$105 mol L 1) in 2 NaOH 25 50 100 150 225 and

300 mL of methanol or DMF were added After each step of the

organic solvent addition the absorption spectra were measured

Additionally the same amounts of water were added to the dye

solution Since the shape of the absorption band did notchange and

its position was shifted less than 200 cm1 it was concluded that

the dilution of the alkaline dye solution could not be the reason of

the observed spectral changes

The measurements of the ionic strength in 1047298uence the dye solution

(005 g L 1) in water containing 002 NaOH was mixed with the

dye solution (005 g L 1) in NaCl solution (362 mol L 1) containing

002 NaOH in the ratio 10 31 11 13 and 01

3 Results and discussion

31 The effect of the dye concentration

UV evis spectra of aqueous solution of IQNB were measured in

concentration range 106$105e 212$104 mol L 1 at four pH

values The sample spectra are shown in Fig 1aec At pH 55 the

dye exists in the solution as a salt (perchlorate) since this pH value

is below the pKa of the dye which equals to 727 [19] The band

corresponding to perchlorate is located at 24721 cm1 (spectra not

shown) In this case any spectral changes with increasing dye

concentration cannot be observed After pH exceeds the pKa value

of the dye dissociation of hydroxyl group occurs and the dye exists

as a merocyanine In UV e

vis spectrum the band corresponding tosalt disappears and the new band appears at 20904 cm1 At pH

115 the band attributed to merocyanine ful1047297ls the LamberteBeer

law up to concentration 317$105 mol L 1 Therefore one can

admit the dye exists as a monomer However the further increase

in the dye concentration leads to a slight bathochromic (red) shift

(Fig 1a)

At pH 135 the increase in the dye concentration causes a

distinct red shift (Fig 1b) It results from the decrease in the in-

tensity of the monomer band (the band at 20904 cm1) which is

accompanied by the rise in the intensity of the new low-energy

band (the band at 18408 cm1) Since the new band appears as

the concentration of the dye increases it can be attributed to the

aggregate If the solution is even more alkaline (at pH 139) the

Scheme 1 The structures of 7H-indolo[12-a]quinolinium dyes


7232019 1-s20-S0022286015004354-main



























N


N

T frac14 0404









phenomenon










aM Aa (1)




K frac14 frac12 Aa

frac12M a (2)







a (4)



obtaining


and












An


n


(7)


n

is the







7232019 1-s20-S0022286015004354-main


and ε Aan



εM n
















ε

Aan

frac14 A

n

frac12D (8)


An


n

thorn X $ε

Aan

(9)

where X $ε Aan



Aan

frac14 ε Aan

=a
















5$105 (2) 8$105 (3) 12$104 (4) path length 1 cm



(2) 244 (3) 476 (4) 698 (5) 1011 (6) 1304 (7)




7232019 1-s20-S0022286015004354-main





2

frac12M 2 (10)












(Fig 5)







aggregates







































































long molecular axis







Table 1


pH 115 135 139


a 204 197 194 191 203 192 190

r 0979 0996 0998 0979 0970 0995 0999

na 6 7 7 6 6 8 7

K (L mol1) 947 3836 932 167$105 122$105 225$104 147$104

a



7232019 1-s20-S0022286015004354-main

































































Xe





















7232019 1-s20-S0022286015004354-main








gregation tendency






















4 Conclusion









References

































7232019 1-s20-S0022286015004354-main














e2765



















e90





7232019 1-s20-S0022286015004354-main



























N


N

T frac14 0404









phenomenon










aM Aa (1)




K frac14 frac12 Aa

frac12M a (2)







a (4)



obtaining


and












An


n


(7)


n

is the







7232019 1-s20-S0022286015004354-main


and ε Aan



εM n
















ε

Aan

frac14 A

n

frac12D (8)


An


n

thorn X $ε

Aan

(9)

where X $ε Aan



Aan

frac14 ε Aan

=a
















5$105 (2) 8$105 (3) 12$104 (4) path length 1 cm



(2) 244 (3) 476 (4) 698 (5) 1011 (6) 1304 (7)




7232019 1-s20-S0022286015004354-main





2

frac12M 2 (10)












(Fig 5)







aggregates







































































long molecular axis







Table 1


pH 115 135 139


a 204 197 194 191 203 192 190

r 0979 0996 0998 0979 0970 0995 0999

na 6 7 7 6 6 8 7

K (L mol1) 947 3836 932 167$105 122$105 225$104 147$104

a



7232019 1-s20-S0022286015004354-main

































































Xe





















7232019 1-s20-S0022286015004354-main








gregation tendency






















4 Conclusion









References

































7232019 1-s20-S0022286015004354-main














e2765



















e90





7232019 1-s20-S0022286015004354-main


and ε Aan



εM n
















ε

Aan

frac14 A

n

frac12D (8)


An


n

thorn X $ε

Aan

(9)

where X $ε Aan



Aan

frac14 ε Aan

=a
















5$105 (2) 8$105 (3) 12$104 (4) path length 1 cm



(2) 244 (3) 476 (4) 698 (5) 1011 (6) 1304 (7)




7232019 1-s20-S0022286015004354-main





2

frac12M 2 (10)












(Fig 5)







aggregates







































































long molecular axis







Table 1


pH 115 135 139


a 204 197 194 191 203 192 190

r 0979 0996 0998 0979 0970 0995 0999

na 6 7 7 6 6 8 7

K (L mol1) 947 3836 932 167$105 122$105 225$104 147$104

a



7232019 1-s20-S0022286015004354-main

































































Xe





















7232019 1-s20-S0022286015004354-main








gregation tendency






















4 Conclusion









References

































7232019 1-s20-S0022286015004354-main














e2765



















e90





7232019 1-s20-S0022286015004354-main





2

frac12M 2 (10)












(Fig 5)







aggregates







































































long molecular axis







Table 1


pH 115 135 139


a 204 197 194 191 203 192 190

r 0979 0996 0998 0979 0970 0995 0999

na 6 7 7 6 6 8 7

K (L mol1) 947 3836 932 167$105 122$105 225$104 147$104

a



7232019 1-s20-S0022286015004354-main

































































Xe





















7232019 1-s20-S0022286015004354-main








gregation tendency






















4 Conclusion









References

































7232019 1-s20-S0022286015004354-main














e2765



















e90





7232019 1-s20-S0022286015004354-main

































































Xe





















7232019 1-s20-S0022286015004354-main








gregation tendency






















4 Conclusion









References

































7232019 1-s20-S0022286015004354-main














e2765



















e90





7232019 1-s20-S0022286015004354-main








gregation tendency






















4 Conclusion









References

































7232019 1-s20-S0022286015004354-main














e2765



















e90





7232019 1-s20-S0022286015004354-main














e2765



















e90





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