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Chapter-V Fluorescence quenching of Anthracene by PF

5. Studies on Quenching of fluorescence of Anthracene and

Proflavin Hemisulphate (PF) and fluorimetric detection of (PF).

5.1: Fluorescence Resonance Energy Transfer between Anthracene and

Proflavin Hemisulphate and analytical application:

The photophysical studies given in Chapter-III indicated that the highly

fluorescent anthracene in micellar solution can interact with proflavin

hemisulphate used as a drug and undergo fluorescence resonance energy

transfer. The absorption results of (PF) and emission results of anthracene in

micellar solutions were examined to obtain suitable analytical correlation.

Fig.5.1 shows significant overlap between fluorescence spectrum of anthracene

and excitation (absorption) spectrum of proflavin hemisulphate. This indicates

that excited athracene molecule can transfer excitation energy to ground state

PF molecule. In addition to this as the absorption spectra of anthracene and PF

shown in Fig. 5.2 are widely separated, the selective excitation of one in

presence of other is possible. The experiment indicated that the PF has

negligible florescence when the radiation of wavelength corresponding to

excitation energy of anthracene was used.

Fig.5.1. Region of Integral overlap between fluorescence spectrum of anthracene (A) and excitation spectrum of PF (B) in SDS micellar solution

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The details of the experiment set to study quenching of fluorescence of

anthracene by PF are given in the Table.5.1. The quenching experiments were

performed in homogenous and heterogeneous media using ethanol and micellar

surfactant solutions of SDS, CTAB and Brij-35 respectively. The concentration

of micellar solution was decided from the studies of effect of concentration of

surfactant on fluorescence intensity of anthracene. Figure 5.3 indicates effect of

concentration of SDS solution on fluorescence intensity of anthracene.

Fig.5.2. Absorption Spectra of Anthracene (A) and PF (B) in Ethanol solution

Table-5.1. Experimental set for fluorescence quenching studies of anthracene by Prolflavin hemisulphate Sr. No

Vol. of 8x10-5 mol dm-3 Anthracene

ml

Vol. of 2x10-5 mol dm-3 PF

ml

ml of SDS/Ethanol Brij-35/CTAB/

ml

Total Volume

Ml

Conc. of Anthracene xs105

Mixture mol dm-3

Conc. Of riboflavin in Mixture [RF] 106

mol dm-3

1 2 00 08 00

2 2 1.0 07 2.0

3 2 2.0 06 4.0

4 2 3.0 05 6.0

5 2 4.0 04 8.0

6 2 5.0 03 10

7 2 6.0 02 12

8 2 7.0 01

10

1.6x

14

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Figure 5.3 shows that intensity of fluorescence increases with

concentration and saturates at higher concentration. From the figure critical

micellar concentration was obtained and concentration of SDS solution above

CMC was used to prepare solution of donor-acceptor pair.

The repeated studies performed on the fluorescence quenching helped us

to decide the suitable concentration of PF and anthracene for quenching

experiments. The concentration of anthracene was kept constant to 1.6 x 10-5

mole dm-3 while that of PF was varied in the range from 2x10-6 to 16 x10-6 mol

dm-3 as is mentioned in the Table.5.1. The quenching experiments were

performed in two sets using SDS solution of concentration below CMC and

other set in which SDS solution of concentration of above CMC.

Fig.5. 3. Effect of surfactant concentration on fluorescence of anthracene

The solubility of anthracene in SDS below CMC is less. Hence its

fluorescence is completely quenched by water soluble PF and gradual

quenching of anthracene fluorescence by PF is not seen in Fig.5.4. The other

set of experiment with similar concentration of anthracene and PF in Ethanol

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and in micellar solutions above CMC of SDS and CTAB produced quenching

of anthracene fluorescence up to satisfaction. Fig.5.4 shows the fluorescence of

anthracene with and without PF in SDS solution of concentration 2x10-3 mol

dm-3 which is below its CMC the gradual quenching of anthracene fluorescence

by PH is not seen. Figure 5.5, 5.6shows the fluorescence quenching results of

anthracene with and without PF in ethanol, SDS solution of 2x10-2 mol dm-3,

concentration respectively [1,2]. The small addition of PF decreases the

fluorescence intensity of annthracene and simultaneously exhibits its broad

structureless band peaking at 506 nm. The successive addition of PF in the

solution decreases the fluorescence of anthracene gradually and that of PF is

enhanced.

Fig.5.4. Quenching of Fluorescence of 1x10-4mol dm-3 anthracene in SDS solution (A) by PF concentration 1x10-7 moldm-3(B) to 6x10-7moldm-3(G)

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Fig. 5.5. Quenching Fluorescence of anthracene (A) 1.6x10-5 by PF concentration: 2 x10-6 mol dm-3 (B) to 10 x10-6 mol dm-3 (F) in ethanol solution

Fig.5.6. Quenching Fluorescence of anthracene (A) 1.6x10-5 by PF concentration: [2 x10-6 mol dm-3 (B) to 14 x10-6 mol dm-3 (H)] in SDS micellar solution

113


The singlet excited anthracene molecules are short lived species [3,4]

and before returning to the ground state undergo Intersystem crossing (ISC),

Internal conversion (IC), fluorescence (F) and enter in bimolecular

fluorescence quenching with ground state PF after FRET. Following

mechanism is proposed and kinetics of quenching process is discussed by

applying steady state approximation.

5.2. Kinetics of Quenching of fluorescence of anthracene by proflavin

hemisulphate in different media.

The fluorescence quenching results fit into well known Stern-Volmer equation.

From the equation no.1 [5-9]. The plot of FF0 Vs [PF] obtained on fluorescence

quenching measurements in different surfactant micellar solution and ethanol

are shown in Fig.5.7. The linearity of plot indicates validity of stern-Volmer

equation. Hence following mechanism similar to Perylene-Riboflavin system is

proposed.

[anthracene]s+ hυo

Ia

Process Rate

[anthracene]*S Excitation Ia

[anthracene]*S

kISC[anthracene]*T

1ISC kisc

1

[anthracene]*S1

[anthracene]*S 1

kIC[anthracene]S o IC kIC

[anthracene]*S1

[anthracene]*S 1

kf[anthracene]S

o+ hυf

Acceptor Fluorescence

kf[anthracene]*S1

[anthracene]*S1+ [PF]S

kq

[anthracene]So+ [PF]*

Quenching Process

kq [anthracene]*S1o

[PF]S o

(Non rediative Energy Transfer)

1

Fluorescence Acceptor Fluorescene

Step

where Ia intensity of radiation absorbed, kIC and kISC are rate constant for

internal conversion and intersystem crossing process.

The experiments on fluorescence quenching of anthracene by PF were

also performed in CTAB and Brij-35 micellar solution shown in Fig.5.8 and

Fig.5.9. The experiments performed have not exhibited the gradual quenching

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of anthracene fluorescence and sensitization of PF fluorescence in Brij-35

micellar solution. However, satisfactory spectral results obtained in CTAB

micellar solution are presented Fig.5.8.

Fig. 5. 7. Plot of FF0 versus [PF] in aqueous SDS (Δ) (2x10-2mol dm-3) CTAB ( ) (2 x10-3mol dm-3), Brij-35 (x) (2x10-4 mol dm-3) Ethanol (♦) solution.

Fig.5 .8.Quenching Fluorescence of anthracene (A) 1.6x10-5 by PF concentration [2 x10-6 mol dm-3(B) to 14 x10-6 mol dm-3(H)] in CTAB micellar solution

115


Fig. 5.9: Quenching Fluorescence of 1.6x10-5 mol dm-3 anthracene (A) by PF concentration [2 x10-6 mol dm-3 (B) to 12 x10-6 mol dm-3(G)] in Brij-35 micellar solution

The quenching results found to fit in to stern-Volmer relation. Fig.5.7. shows

plot of FF0 Vs [PF] for the results obtained in Ethanol, SDS CTAB and Brij-

35 micellar solution. The plots are straight lines with intercept one on Y-axis

and indicate validity of Stern-Volmer relation for bimolecular fluorescence

quenching of anthracene by PF in Ethanol, SDS CTAB and Brij-35 solutions.

The values of Stern-Volmer constant Ksv from the slope of lines and quenching

rate constant calculated from the equations are given in Table.2

The estimated value of the quenching rate constant (kq) is determined to

be 9.221x 1012 6.935 x 1012 and 7.9918x1012 mol-1dm3 s-1 in Ethanol and SDS,

CTAB micellar media. The value of kq falls in the range (~1011-1012 mol-1dm3

s-1) reported earlier for similar type FRET studies and is order of magnitude

higher agreement with values reported for a normal diffusion controlled

quenching process [10-12]. The KSV and kq values also suggest that the

dominant mechanism of the fluorescence quenching is the resonance energy

transfer through long range dipole-dipole interaction rather than the simple

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diffusion dominated collision processes between the excited donor and the

ground state acceptor molecules

Table. 5.2. Structural and kinetics parameters of donor and acceptor. Solvents Life

Time (ns)

Forster Radius (R0)Å

10-4 Ksv

mol-1 dm3 s Distance between Donor and Acceptor

(r )nm

Efficiency of energy transfer

10-12 kq

mol dm-3 s-1

SDS 4.5 19.3 3.1692 2.39 0.1573 6.935

CTAB 4.9 18.1 3.9168 2.75 0.1834 7.991

Brij-35 4.4 26.4 2.3311 3.53 0.1125 5.203

Ethanol 5.0 37.9 4.6296 4.28 0.2006 9.221

5.3. Efficiency of Energy transfer processes:

According to the Förster theory, the efficiency of energy transfer (η)

was calculated by the equation 7 [13-18]. The estimated values of (η) are

graphically presented as function of concentration of PF in Fig.5.10. It is seen that

efficiency energy transfer increases with increasing concentration of PF.

The value of η is shown to depends upon the distance between donor

and acceptor molecules (r) and critical energy transfer distance R0 at which

efficiency of energy transfer is 50% given by equation 8 [19-21]. The value of r

found to be 2.396 Å in SDS micellar solution. This value is too far to transfer

electron from anthracene to PF molecule but satisfy the conditions of

fluorescence resonance energy transfer [22-23]. The estimated values of R0 are

19.34 Ǻ and SDS, 37.9 Ǻ in Ethanol. The value of R0 is less than ≈ 50 Ǻ are an

indication of efficiency of energy transfer between the donor-acceptor pair. The

Ro value < 50 Å indicates efficient energy transfer between anthracene and

PF[24-25]. In SDS solution the anthracene is present in micelle core and PF

attached with hydrophilic region of micelle are at distance 2.396 nm required

for efficient energy transfer as shown in Fig. 5. 11.

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Fig. 5. 10. Efficiency of energy transfer between anthracene and PF

Fig. 5. 11. Schematic figure shows the anthracene and PF in Micellar solution

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5.4. Effect of foreign substances and method of analysis:

This study was carried out to detect possible interferences of other

species that normally accompany the analyte in sample. A mixture of

anthracene and PF containing 1.6x10-5 mol dm-3 and 8x10-6 mol dm-3 and a

known amount of the foreign species under study was interested into the

system. The signal was recorded, and if the error caused was at least ±5% in

comparison with another solution that did not contain the foreign, the foreign

species was reduced progressively until the interference ceased.

The study was focused principally on anions, cations. It is interesting to

point out the selectivity that the proposed system shows that the levels of the

species under study that commonly can be found in real samples are in all case,

lower than the tolerated ones by the system. The substances tested and the

tolerance ratios are summarized in Table.3.

5.5. Estimation of PF from pharmaceutical Sample:

Fluorescence Technique was used for estimation of proflavine

hemisulphate (PF) from ointment Lorexane made, commercial available in the

market by Virbac Animal Health India, Pvt. Ltd appropriate amount of sample

diluted with SDS solution and were assayed by the standard procedure. A

series of standard solution of Anthracene-PF FRET system under investigation

were prepared. The compositions were kept same for standard solution as well

as pharmaceutical sample. For every solution fluorescence intensities were

measured and the calibration curve plotted. In similar way the Fluorescence

Intensity of pharmaceutical samples is obtained and F0/F calculated from the

calibration curve. For the accuracy of the proposed method three different

concentrations of sample solutions were used in quenching experiment to

determine the values of F0/F in presence of anthracene. The amount of PF

present in the ointment was calculated and given in the Table 4. along with its

real composition used during formulation. From these results to confirm the

reliability of the results, standard deviation and relative standard deviation in

present were calculated and given in the same Table 4. The negligibly small

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value of RSD i.e. 2.15% indicates that the proposed method gives accurate

results.

Table 5.3: Effect of interfering substances on fluorescence of anthracene-proflavin hemisulphate system in SDS micellar solution Interfering substance

Concentration (mg .L-1)

(%)Change in Fluorescence Intensity at fixed wavelength λex = 360nm & λem = 405 nm

Al+3

Ba+2

Mn+2

Bi+2

Zn+2

EDTA

CuCl2

0.01

0.19

0.01

0.058

11.53

0.096

0.002

1.25

0.25

2.75

3.00

4.5

2.015

2.25

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Table. 5. 4: Composition of Proflavin Hemisulphate from Pharmaceutical Sample (experimental and real sample)

Name of

Sample

Amount of Proflavin

Hemisulphate (%)

Certified value Found value

(experimental )

RSD %

SD

Lorexane

Virbac

Animal

Health India,

Pvt. Ltd

0.1 W/W

0.082 W/W

2.15

0.0017

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