5. Studies on Quenching of fluorescence of Anthracene and 5.pdf  Studies on Quenching of fluorescence

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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

    109

  • Chapter-V Fluorescence quenching of Anthracene by PF

    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 xs105Mixture mol dm-3

    Conc. Of riboflavin in Mixture [RF] 106mol 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

    110

  • Chapter-V Fluorescence quenching of Anthracene by PF

    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

    111

  • Chapter-V Fluorescence quenching of Anthracene by PF

    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)

    112

  • Chapter-V Fluorescence quenching of Anthracene by PF

    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

  • Chapter-V Fluorescence quenching of Anthracene by PF

    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+ ho

    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+ hf

    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

    114

  • Chapter-V Fluorescence quenching of Anthracene by PF

    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 () (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

  • Chapter-V Fluorescence quenching of Anthracene by PF

    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