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0022-2313/$ - se

doi:10.1016/j.jlu

�Correspondifax: +918472 2

E-mail addre

(R.S. Kunabenc

Journal of Luminescence 116 (2006) 35–42

www.elsevier.com/locate/jlumin

Analysis of fluorescence quenching of new indole derivative byaniline using Stern–Volmer plots

H.M. Suresh Kumara, R.S. Kunabenchia,�, J.S. Biradarb, N.N. Mathc,J.S. Kadadevarmathc, S.R. Inamdarc

aDepartment of Physics, Gulbarga University, Gulbarga 585 106, IndiabDepartment of Chemistry, Gulbarga University, Gulbarga 585 106, IndiacDepartment of Physics, Karnatak University, Dharwad 580 003, India

Received 13 November 2003; received in revised form 30 October 2004; accepted 29 December 2004

Available online 15 April 2005

Abstract

The fluorescence quenching of 5-methyl-3-phenyl-2-[40-amino-s-triazolo-30-yl] indole-50-hydrazide (MPIH) by aniline

quencher in cyclohexane, benzene, toluene and dioxane solvents has been carried out at room temperature with a view

to understand the quenching mechanisms. The quenching is found to be appreciable and shows positive deviation in the

Stern–Volmer plots. The various quenching parameters have been determined using modified Stern–Volmer equation,

which suggests that the sphere of action static quenching model agrees very well with the experimental results and this

positive deviation is attributed to the static and dynamic quenching. Further, with the use of the finite sink

approximation model, it is concluded that these bimolecular quenching reactions are diffusion limited and the distance

parameter R0 and mutual diffusion co-efficient D are estimated independently.

r 2005 Elsevier B.V. All rights reserved.

PACS: 31.70.Hq; 87.15.Kg; 87.64.Ni

Keywords: Indole; Fluorescence; Quenching; Stern–Volmer plot; Static and dynamic quenching; Diffusion limited

e front matter r 2005 Elsevier B.V. All rights reserve

min.2005.02.012

ng author. Tel.: +918472 245512;

45927/245632.

ss: kunabenchi@yahoo.co.in

hi).

1. Introduction

The fluorescence quenching phenomenon oforganic molecules using external quenchers inliquid phase has been widely studied by variousauthors to understand the nature of bimolecularreactions taking place both under steady-state andtransient conditions [1–6]. This study is very

d.

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CH3

N

H

Ph

N N

N

NH 2

NH NH 2

Fig. 1. Molecular structure of 5-methyl-3-phenyl-2-[40-amino-s-

triazolo-30-yl] indole-50-hydrazide.

H.M. Suresh Kumar et al. / Journal of Luminescence 116 (2006) 35–4236

important in physical, chemical, biological andmedical sciences [6–8]. In bimolecular liquidsystems, the fluorescence intensity is hindereddue to several mechanisms, such as static anddynamic quenching, excimer and exciplex forma-tion, charge transfer process, etc. [2–4,8,9]. Therole of fluorescence quenching can be studiedexperimentally by determining quenching rateparameters using Stern–Volmer (S–V) plots thatare drawn in accordance with the S–V equation:

I0=I ¼ 1þ KSV½Q� (1)

and

t0=t ¼ 1þ K 0SV½Q�, (2)

where I0 and t0 are the fluorescence intensity andfluorescence lifetime, I and t are the fluorescenceintensity and fluorescence lifetime in the absenceand presence of the quencher concentration [Q],respectively, and KSV (K 0

SV) is the S–V constant. Inmany cases, the S–V plots were found to be linear,in which the quenching mechanism is mainly dueto the dynamic process, where diffusion process isthe dominant one [10,11]. In some cases, theexperimental results show positive deviation fromthe linear S–V relation [1–3,12–14]. This findingmay be due to one of the above processes otherthan or along with diffusion process. Apart fromthis, the polarity of the solvent medium and therange of quencher concentration are also expectedto play a part in this medium [15].In the present study, we have studied the steady-

state fluorescence quenching of the newly synthe-sized indole derivative 5-methyl-3-phenyl-2-[40-amino-s-triazolo-30-yl] indole-50-hydrazide[MPIH]at room temperature using aniline as a quencher incyclohexane, dioxane, benzene and toluene sol-vents, and also by the transient method in toluenesolvent to understand the nature of the quenchingmechanism.

2. Experimental details

The solute, MPIH was synthesized in ourlaboratory using standard methods [16,17] andwas purified by double re-crystallization usingacetone-ethyl alcohol solvents. The molecular

structure of MPIH is shown in Fig. 1. Thespectroscopic grade solvents (S.D. Fine ChemicalsLtd.) have been used without further purificationto prepare the solutions. However, the purity ofthe solvents was checked by recording the back-ground fluorescence. Spectroscopic grade anilinesolvent has been used as a quencher and it wasdouble distilled before use.Absorption spectra were recorded in different

solvents using UV–visible absorption spectrophot-ometer (Hitachi model 150-20) at the concentra-tion of 1� 10�5mol dm�3. The steady-statefluorescence spectra were recorded for the sameconcentration by exciting the sample at 320 nmradiation corresponding to the longer absorptionpeak using fluorescence spectrophotometer(Hitachi model F-2000) in all the solvents byvarying the quencher concentration from 0.00 to0.10mol dm�3. The fluorescence decays of thesolute were recorded without and with quencherin toluene solvent using picosecond time correlatedsingle photon counting technique (TCSPC) (Mod-el 5000U, IBH, UK). The third harmonic picose-cond laser pulse of wavelength 310 nm, derivedfrom the mode locked Ti-Sapphire laser (ModelSpectra Physics, Tsunami) pumped by Nd-YVO4

laser was used as an excitation source. The decayswere measured at 390 nm corresponding to fluor-escence maxima. The data analysis was accom-plished by the software DAS-6 (IBH) based on thedeconvolution technique using iterative nonlinearleast square methods. The quality of the fit isidentified by the reduced chi-square value,weighted residuals and the autocorrelation func-tion of the residuals.

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0

1

2

3

4

0 5 10 15 20

τ 1 = 0.56 ns, a1 = 0.18 τ 2 = 2.34 ns, a2 = 0.82

τ ave = 2.02ns, 2� = 1.2

log

(cou

nts)

Time (ns)

Fig. 3. Fluorescence decay profile of MPIH in toluene solvent

at the concentration of 1� 10�5mol dm�3.

0.00 0.02 0.04 0.06 0.08 0.100

2

4

6

8

10

(I0

/I)

or (

τ τ 0

/τ τ )

Dioxane

Toluene

Benzene

Cyclohexane

[Q]

I0 / I vs [Q]

τ0 / τ τ vs [Q]

Fig. 4. S–V plot of I0=I against [Q] in different solvents and

t0=t against [Q] in toluene solvent with aniline quencher.

H.M. Suresh Kumar et al. / Journal of Luminescence 116 (2006) 35–42 37

3. Results and discussion

The steady-state fluorescence intensities I0 and I

were measured in the absence and presence ofquencher, respectively, in cyclohexane, dioxane,benzene and toluene solvents at fixed MPIHconcentration. The typical absorption and fluor-escence spectra in toluene solvent with differentquencher concentrations are shown in Fig. 2.Further, the fluorescence lifetimes t0 and trepresenting without and with aniline quencherwere measured in toluene solvent. The typicalfluorescence decay profile in toluene solvent with-out quencher is shown in Fig. 3. It is observed thatthe fluorescence decay was fitted to bi-exponentialwith lifetime values t1 and t2: However, theaverage lifetime, /tfS ¼ t1a1+t2a2 (where a1and a2 are the amplitudes) is considered inestimating the quenching parameters, since theaverage lifetime agrees closely with the lifetimevalues of the other indole derivatives [2,11].The S–V plots of I0=I against [Q] in different

solvents and t0=t against [Q] in toluene solventwith aniline quencher were drawn and are shownin Fig. 4. It is observed that in both cases, the S–Vplots are nonlinear, showing positive deviation.Similar experimental results for other indolederivatives were observed by others [2,6,12,18].From the S–V plot, it may be concluded that thequenching is not purely collisional and may be due

0.00

0.10

0.00

0.10

300 400

Wavelength, nm

500 nm

280 300 325 350

Inte

nsity

(a.

u.)

Opt

ical

Den

sity

(a.

u.)

(b) (a)

Fig. 2. (a) Absorption spectra, (b) fluorescence spectra of

MPIH in toluene at the concentration of 1� 10�5mol dm�3

with quencher concentration of aniline from 0.00 to

0.10mol dm�3.

to the formation of either the ground statecomplex or static quenching process [1–3,12,13].However, the maxima of the longer wavelengthabsorption band and fluorescence maxima of theMPIH do not change in the presence of quencherin all the solvents and also, no additional band wasobserved at a longer wavelength. These factorsruled out the possibility of ground state complexformation [2,3,19]. Thus, the analysis of the datawas carried out by employing the ‘‘sphere of actionstatic quenching model’’. According to this model,the deviation from linear S–V plots is due to thefact that only a certain fraction ‘W ’ (in case ofsteady-state condition) or W 0 (in case of transient)of the excited state is actually quenched bycollisional mechanism. This static quenching was

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H.M. Suresh Kumar et al. / Journal of Luminescence 116 (2006) 35–4238

explained by introducing an additional factors W

and W 0 in the linear S–V Eqs. (1) and (2),respectively [7]

I0=I ¼ ð1þ KSV½Q�Þ=W , (3)

t0=t ¼ ð1þ K 0SV½Q�Þ=W 0. (4)

In such cases, some molecules in the excitedstate, the fraction of which is (1� W ), or (1� W 0)are de-activated almost instantaneously after beingformed, because a quencher molecule happens tobe randomly positioned in the proximity at thetime the molecules are excited and interacts verystrongly with them. Thus, the fraction W (W 0)decreases from unity in contrast to the simple S–VEqs. (1) and (2) where W ¼ 1 and W 0 ¼ 1;respectively. Hence, the instantaneous or staticquenching occurs if the quencher molecule is verynear to or in contact with the fluorescent moleculeat the moment of excitation. The factor W (W 0) inthe modified S–V Eq. (3), (4) is approximatelyequal to exp (�V [Q]), (exp (�V0[Q])), where V

(V 0) is the static quenching constant and itrepresents an active volume element surroundingthe excited solute molecule.Frank and Wawilow [20] have suggested that

the instantaneous quenching results at the in-stances in a randomly distributed system, when aquencher happens to reside within a ‘‘sphere ofaction’’ with a volume of V/N0 (V0/N0) and radius r

0.0 0.2 0.40

5

10

15

20

25

30

35

40

(1-

I/I 0)

/[Q

] or

(1-

τ /τ

τ /τ

0)/[

Q]

[1-(I / I0)] / [Q] vs I / I0

[1–(τ / τ0 )] / [Q] vs τ τ / τ0

I

Fig. 5. Plot of [1�(I/I0)]/[Q] against I=I0 in different solvents and [1�

(r0) (kinetic distance), i.e. V=N 0 ¼ 4pr3=3 (4pr03/3)surrounding a solute molecule at the time ofexcitation. On excitation of the solute molecule, aquencher molecule, which is already within thisvolume, will be able to quench the fluorescencewithout the need for a diffusion-controlled colli-sional interaction. The probability of the quencherbeing within this volume at the time of excitationdepends on the volume V (V 0) and on thequencher concentration [Q]. Hence, it is mean-ingful to re-write Eqs. (3) and (4), respectively, asfollows:

½1� ðI=I0Þ�=½Q� ¼ KSVðI=I0Þ þ ð1� W Þ=½Q�, (5)

½1� ðt=t0Þ�=½Q� ¼ K 0SVðt=t0Þ þ ð1� W 0Þ=½Q�. (6)

The modified S–V plots [1�(I/I0)]/[Q] againstI=I0 for steady state in different solvents, and[1�(t/t0)]/[Q] against (t=t0) for transient case intoluene solvent only, were drawn and found to belinear as shown in Fig. 5. The S–V quenchingconstant KSV (K 0

SV) were obtained using least-square fit method by determining the slopes.The quenching rate parameters kqð¼ KSV=t0Þ andk0

qð¼ K 0SV=t0Þ were calculated using the experi-

mentally determined KSV (K 0SV) and t0 values. The

intercepts of least-square fit lines of Fig. 5 areequal to (1�W)/[Q] and (1�W0)/[Q]. From theseintercepts, the values of W (W 0) were calculatedfor each quencher concentration and the range of

0.6 0.8 1.0

Cyclohexane Toluene Benzene Dioxane

/ I0

(t/t0)]/[Q] against t=t0 in toluene solvent with aniline quencher.

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Table 1

The values of dynamic quenching constant KSV, quenching rate parameter kq, range of W, static quenching constant V and radius of

the sphere of action (kinetic distance) r

Solvent KSV (M�1) kq� 10�9 (M�1 s�1) Range of W V (M�1) r (A)

Cyclohexane 45.02 22.29 0.67–0.92 4.26 11.91

Dioxane 10.38 05.14 0.77–0.95 2.65 10.21

Benzene 12.76 06.32 0.66–0.93 4.47 12.12

Toluene 25.72 12.73 0.72–0.94 3.25 10.93a1.13 0.56 0.64–0.93 4.37 11.97

RS ¼ 4:13 (A; RAniline ¼ 2:84 (A; tav ¼ 2:02 ns:aData from lifetime measurements.

H.M. Suresh Kumar et al. / Journal of Luminescence 116 (2006) 35–42 39

W is given in Table 1. Using the values of W (W 0),the static quenching constant V (V 0) was obtainedby least-square fit method using the equation W ¼

expð2V ½Q�Þ ðW 0 ¼ expð2V 0½Q�ÞÞ and in turn thekinetic distance r (r0), i.e. ‘‘radius of sphere ofaction’’ was determined by least-square fit methodaccording to the equation V/N0 ¼ 4pr3/3 (V0/

N0 ¼ 4pr03/3). All these data are collated in Table1. It is observed from Table 1 that the values ofKSV are rather larger compared with V in all thesolvents, which explains the lack of absorptionchange on addition of the quencher [3].The radii of the solute, RS; and that of the

quencher, RQ; molecules were determined byadding the atomic volumes of all the atomsconstituting the molecule as suggested by Edward[21] and are given at the bottom of Table 1. Thesum of the molecular radii R of the solute andquencher is determined and is called as encounterdistance or contact distance or reactive distance.This value of R is then compared with the value ofkinetic distance (r) to verify whether the reaction isdue to sphere of action or not. According to Andreet al. [1] and Zeng et al. [9], if the distance betweenthe quencher molecule and excited molecule liesbetween the encounter distance R (i.e. the sum ofthe molecular radii of the interacting molecules)and the kinetic distance r (i.e. the radius of sphereof action) the static effect takes place especially inthe case of steady-state experiments irrespective ofground state complex formations provided thereactions are limited by diffusion. From Table 1, itis observed that the values of kinetic distance r (r0)are higher than the encounter distance R in all thesolvents indicating the sphere of action static

quenching model agrees well in our case also[10–14].Further, to find out, whether the reactions are

diffusion limited or not, we invoke the finite sinkapproximation model for steady state which helpsus to estimate independently the mutual diffusioncoefficient, D; distance parameter, R0; and activa-tion energy controlled rate constant, ka: Themodified S–V equation [9]

K�1SV ¼ ðK0

SV�1

�ð2pN 0Þ

1=3

4pN 0Dt0½Q�1=3, (7)

where

K0SV ¼

4pN 0DRt0ka

4pN 0DR þ ka(8)

of finite sink approximation model is used todetermine values of D; R0 and ka: For efficientquenching processes, the value KSV is oftenobserved to increase with [Q] [9]. Hence, the valuesof KSV were determined at each quencher con-centration in all the solvents. The graphs K�1

SV

against [Q]1/3 were plotted as shown in Fig. 6 usingEq. (7) and were found to be linear in all the cases.Using least-square fit method, the values of mutualdiffusion coefficient D and K0

SV (S–V constant at[Q] ¼ 0) were determined by measuring the slopesand intercepts. The distance parameter R0 wascalculated by using these values according to theequation K0

SV ¼ 4pN 0DR0t0 and these values aregiven in the Table 2. The activation energycontrolled rate constant ka½¼ 4pN 0DR=ðR=R0 �

1Þ� was estimated by considering the values ofdistance parameter R0 and encounter distance R:This ka value can be determined only when R0oR:

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0.0 0.1 0.2 0.3 0.4 0.50.00

0.02

0.04

0.06

0.08

0.10 Dioxane Benzene Toluene Cyclohexane

1− S

VK

[Q]1/3

[Q]1/3

0.0 0.1 0.2 0.3 0.4 0.50.0

0.1

0.2

0.3

1− S

VK

Toluene

(a)

(b)

Fig. 6. Plots of (a) K�1SV against [Q]1/3 determined from fluorescence intensities in different solvents, (b) K�1

SV against [Q]1/3 determined

from lifetime measurements in toluene solvent with aniline quencher.

Table 2

The values of K0SV (steady state quenching constant at [Q] ¼ 0), mutual diffusion co-efficient D; distance parameter R0; 4pN0DR0,

activation energy controlled rate constant ka

Solvent K0SV (M�1) D� 105 (cm2 s�1) R0 (A) 4pN0DR0 � 10�9 (M�1 s�1) ka� 1015 (M�1 s�1)

Cyclohexane 35.11 3.19 7.20 17.38 —

Dioxane 10.10 1.24 4.91 04.61 1.26D

Benzene 12.34 1.04 7.15 05.63 —

Toluene 22.72 2.83 4.84 10.36 1.20Da3.45 0.36 6.42 01.12 6.15D

Quenching rate constant for diffusion controlled reaction kd ¼ 0:53D � 1015 M�1 s�1:aData from life time measurements.

H.M. Suresh Kumar et al. / Journal of Luminescence 116 (2006) 35–4240

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H.M. Suresh Kumar et al. / Journal of Luminescence 116 (2006) 35–42 41

According to Zeng et al. [9], if ka is greater thankdð¼ 4pN 0DRÞ; then the reactions are said to bediffusion limited. Here, the values of both ka andkd are expressed in terms of D (mutual diffusionco-efficient, determined by Stokes–Einstein rela-tion) because D is same in both the cases and thekdð¼ 4pN 0DRÞ value is given at the bottom of theTable 2. It is observed that ka is greater than kd indioxane and toluene solvents. It suggests that theactivation process is more predominant in thequenching mechanism than the diffusion process[13,18]. Thus, it can be predicted that thesereactions are said to be diffusion limited [9]. SinceR0 is greater than R in case of cyclohexane andbenzene, ka cannot be determined. But accordingto Joshi et al. [18], the bimolecular quenchingreactions are said to be diffusion limited ifkq44pN 0DR0: Hence, we determined 4pN 0DR0

and it is observed that kq44pN 0DR0; which isexpected to say that the reactions are diffusionlimited. Further, a positive deviation in the S–Vplot is likely when both static and dynamicquenching processes occur simultaneously.From Table 3, it is important to note that the

values of mutual diffusion co-efficient D deter-mined by finite sink approximation are notcomparable with the values of D determined byStokes–Einstein relation in all solvents. Similarly,R0 and R values determined by finite sinkapproximation and Edward’s empirical relationare comparable in benzene and cyclohexane butnot in dioxane and toluene. In fact, similardiscrepancies have been reported [9,13,18] in somebimolecular quenching reactions. This may be due

Table 3

The values of mutual diffusion co-efficients Da and Db, distance

parameter R0 and encounter distance R

Solvent Da� 105 (cm2 s�1) Db

� 105 (cm2 s�1) R0 (A)

Cyclohexane 2.33 3.19 7.23

Dioxane 1.75 1.24 4.91

Benzene 3.53 1.04 7.15

Toluene 3.78 2.83 4.84

aDiffusion co-efficient determined from Stokes–Einstein

relation.bDiffusion co-efficient determined from finite sink relation.

R ¼ ðRS þ RQÞ ¼ 4:13þ 2:84 ¼ 6:97 (A:

to the uncertainties in the values of the adjustableparameter ‘a’ in the Stokes–Einstein relation andthe approximation in the values of the atomicvolume in the Edward’s empirical relation. Hence,it is reasonable to conclude that the finite sinkapproximation model is valid in recovering theparameters D and R0ðRÞ:Specific conclusions emerged from the presented

work are: (a) the Stern–Volmer plots show positivedeviations leading to high values of kq; (b) thevalues of ka are greater than kd in dioxane andtoluene and kq is greater than 4pN 0DR0 incyclohexane and benzene and (c) finite sinkapproximation model is valid in recovering theparameters D and R0ðRÞ: Hence, it may beconcluded that both static and dynamic (transient)quenching processes are playing a part in thissystem.

Acknowledgements

The authors wish to thank Professor P. Natar-ajan and Dr. P. Ramamurthy, National Center forUltrafast Processes, Chennai, for providing thetime-correlated single-photon counting techniquefacility to measure the lifetimes. The authorswould like to thank the technical staff of theUSIC, Karnatak University, Dharwad, for theirhelp in recording the absorption and fluorescencespectra.

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