CHAPTER I Introductionshodhganga.inflibnet.ac.in/bitstream/10603/62725/6/06_chapter_01.pdfCHAPTER...

CHAPTER – I Introduction

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1. Introduction:

Present work is based on photoinduced electron transfer (PET) between

electron donor-acceptor systems. The process involves absorption of UV-

visible photon by one of the component which is specifically fluorescent and

has capability either to donate or accept electron with the molecule of other

component present within the approach of 10 Ao in suitable solvent

environment to get binded. The binding between two molecules of different

components in the excited state results into decrease in fluorescence of excited

molecule. Hence such process involving electron transfer on absorption of UV

-visible photon called PET can be studied by measuring the quenching of

fluorescence of photosensitive component as a function of concentration of

other component which may be an analyte of significance as pharmaceutical,

biological and environmental molecule. The systematic measurement on

intelligently selected appropriate electron donor-acceptor molecules of two

different components helped to develop new analytical methods for detection

as well as to understand molecular interactions. Therefore the subject

fluorescence, PET processes and their applications are reviewed in this chapter.

1.1 Luminescence:

Luminescence is the emission of light from any substance that occurs

from electrically excited state. Stokes (1852) formulated the first law in the

history of luminescence, which states that the wavelength of the luminescence

is greater than the wavelength of the exciting radiation [1]. The term

“Luminescence” was introduced into the literature by Wiedemann (1888). He

also offered the first, although not entirely accurate, defination of luminescence

as the excess emission over and above the thermal emission background [2].

This definition reflected an important property, but it did not distinguish

luminescence from other type of glow that is also excess emission over and

above thermal emission background. Vavilov (1951-52) suggested

complimenting Wiedemann‟s definition by addition the correction of duration

and using the term luminescence for the excess emission over and above the


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thermal emission of a body, if this emission has a duration considerably

exceeding period of light oscillations [1-3]. Thus luminescence is the emission

of cold light in which all or part of absorbed energy is re-emitted in the form of

electromagnetic radiation in the visible or near visible region of the spectrum.

It involves two main steps, the excitation of the electronic system of the

material and the subsequent emission of photons. The examples of

luminescence are the light or glow emitted by a luminous watch dial, fireflies,

glowworms, the lantern fish (Lucerna piscis), Nile fish (dilyxnos), bacteria,

rotten wood, fungus, mushrooms, mollusc, squid, etc. Luminescence contrasts

with incandescence, which is the production of light by heated materials [1-5].

The processes of luminescence are detailed as,

1. Absorption of energy by the electron of the atoms of the absorbing

materials causes the excitation of electron and it jumps from the inner orbits of

the atoms to the outer orbits.

2. When the electrons fall to their original state, a photon of light is

emitted. The interval between the steps may be short (less than 1/10, 000 of a

sec.) or long (many hours). If the interval is short, the process is called

fluorescence and if the interval is long, the process is called phosphorescence.

In either case the light produced is almost always of lesser energy and of longer

wavelength than the exciting light [1, 3-4].

Present studies involve monomer fluorescence and it‟s quenching by

electron transfer hence fluorescence and its aspects are revised.

1.2 Photoluminescence:

Generating luminescence through excitation of a molecule by ultraviolet

or visible light photons is a phenomenon termed photoluminescence. It is

formally divided into two categories as fluorescence and phosphorescence,

depending upon the electronic configuration of the excited state and emission

pathway [6].


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Fig. 1.1.a: Spin configuration of singlet and triplet excited states.

The electronic states of most organic molecules can be divided into singlet state

and triplet state. In singlet state, all electrons in the molecules are spin-paired

while in triplet state, one set of electron spins is unpaired as shown in Fig.1.1.a.

1.3 Fluorescence:

Fluorescence is emission of light from singlet-excited states, in which

the electron in the excited orbital return to the ground state and this spin-

allowed transition occurs rapidly by emission of a photon [4, 7-8]. With a few

rare exceptions, molecules in condensed phase rapidly relax to the lowest

vibrational level of S1. This process, called internal conversion, is nonradiative

and takes place in 10-12

seconds or less. Return to the ground state occurs to a

higher excited vibrational ground-state level, which then quickly reaches

thermal equilibrium. An interesting consequences of emission to a higher

vibrational ground state is that the emission spectrum is typically a mirror

image of the absorption spectrum of the 10 SS . This emission rates of

fluorescence are 108

s-1

, so that a typically fluorescence lifetime is near 10

nanoseconds. Fluorescence spectral data are generally presented as emission

spectra. Emission spectra vary widely and are dependant upon the chemical

structure of the fluorophore and the solvent in which it is dissolved.


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1.3.1 Mechanism of Fluorescence:

On absorption of light the molecule is raised from the ground state G to

the excited state E, the resulting electronic transition is shown in Fig.1.1.b as

per the Frank- Condon principle [9].

A Absorption F Fluorescence T Triplet state

P Phosphorescence

Fig.1.1.b: Electronic transitions

The molecule returns to the ground state by emitting some of its absorbed

energy as fluorescence. An electronic transition due to light absorption is

almost instantaneous (10-15

second) whereas the lifetime of the excited state is

about 10-8

second and therefore the whole process of light absorption and

fluorescence emission takes place in about 10-8

second. Now some of the

absorbed energy is lost partly by collisions with other molecules so that the

emitted energy is less than the energy absorbed from the exciting light.

Fluorescence is thus an emission from a singlet excited state (electrons spin

paired) to ground state where as phosphorescence is emission from a triplet

excited state (electrons unpaired) to ground state. Phosphorescence persists

longer than fluorescence and this persistence are prolonged and the intensity

enhanced by low temperature [7].

1.3.2 Jablonski diagram:

Processes, which occur between the absorption and emission of light,

are usually illustrated by Jablonski diagram shown in Fig. 1.2. The ground, first

and second electronic states are depicted by S0, S1 and S2, respectively. At each

E

G

Excited state

(Singlet)


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electronic energy level, the fluorophores can exist in a number of vibrational

energy levels. Transitions between states are depicted as vertical lines to

illustrate the instantaneous nature of light absorption. According to the Franck-

Condon principle, transitions occur in about 10-15

seconds, a time is too short

for significant displacement of nuclei [5]. The highest probability of the

transition corresponds to the largest overlap between the ground state and

excited-state vibrational wavefunctions.

Fig.1.2: A simplified diagram with absorbance, internal conversion,

fluorescence, intersystem crossing and phosphorescence

1.3.3 Types of Fluorescence: ,

The fluorescence which is observed as long as excitation is in process is

known as steady state or prompt fluorescence. When excitation is cut off, this

fluorescence ceases. However, in some case even after cutting of source of

excitation, the emission of light persists as a glow and is known as delayed

fluorescence.

1. Steady State Fluorescence:

2. Delayed Fluorescence


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1.4 Processes competing with Fluorescence:

1.4.1 Vibrational Relaxation (VR):

Emission spectra are typically independent of the excitation wavelength.

Upon excitation to higher electronic and vibrational levels, the excess energy is

quickly dissipated, leaving the fluorophore in the lowest vibrational levels of

S0. As a consequence, fluorescence from solution, when it occurs, always

involves a transition from the lowest vibrational level of an excited state S1 [4,

10]. Due to vibrational relaxation the fluorescence band for a given electronic

transition is displaced toward lower frequencies or longer wavelengths from the

absorption bands (Stoke‟s effect).

1.4.2 Internal Conversion (IC):

Internal conversion is a non-radiative transition between two electronic

states of the same spin multiplicity. In solution, this process is followed by a

vibrational relaxation towards the lowest vibrational level of the final electronic

state. The excess vibrational energy can be indeed transferred to the solvent

during collisions of the excited molecule with the surrounding solvent

molecules. Thermally relaxed excited molecules transfer energy into

isoenergetic vibrational level of ground state level which follows vibrational

relaxation [11]. Internal conversion from S1 to S0 is also possible but is less

efficient than conversion from S2 to S1, because of the much larger energy gap

between S1 and S0. Therefore, internal conversion from S1 to S0 can compete

with emission of photons (fluorescence) and intersystem crossing to the triplet

state from which emission of photons (phosphorescence) can possibly be

observed.

1.4.3 Intersystem Crossing (ISC):

Intersystem crossing is a non-radiative transition between two

isoenergetic vibrational levels belonging to electronic states of different

multiplicities. For example, an excited molecule in the 0 vibrational level of the

S1 state can move to the isoenergetic vibrational level of the Tn triplet state;


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then vibrational relaxation brings it in to the lowest vibrational level of T1.

Intersystem crossing may be fast enough (10-7

–10-9

s) to compete with other

pathways of de-excitation from S1 (fluorescence and internal conversion

S1 S0) Crossing between states of different multiplicity is forbidden, but spin–

orbit coupling (i.e. coupling between the orbital magnetic moment and the spin

magnetic moment) can be large enough to make it possible. The probability of

intersystem crossing depends on the singlet and triplet states involved. If the

transition S0 S1 is of *n type for instance, intersystem crossing is often

efficient. It should also be noted that the presence of heavy atoms (i.e. whose

atomic number is large, for example Br, Pb) increases spin–orbit coupling and

thus favors intersystem crossing [12].

Once ISC has occurred, the molecule undergoes the usual IC process

and falls to the lowest vibrational level of the first excited triplet state.

Therefore, ISC can compete with fluorescence and thus it decreases the

quantum efficiency of fluorescence. The population of triplet state has

significance in producing delayed fluorescence and phosphorescence, which is

a radiative decay of triplet state molecule to the ground state. Different

pathways for de-excitation of excited molecules are exhibited in Fig. 1.3.

Fig.1.3: De-excitation pathways of excited molecules


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1.5 Structural factors on Fluorescence Measurements:

1.5. a. Effect of electron-withdrawing substituents: (carbonyl and nitro

compounds)

The fluorescence properties of aromatic carbonyl compounds are

complex and often difficult to predict. Many aromatic aldehydes and ketones

(e.g. benzophenone, anthrone, 1- and 2- naphthaldehyde) have a low-lying n–p

excited state and thus exhibits low fluorescence quantum yields, as explained

above and the dominant de-excitation pathway is intersystem crossing.

O O

OO

O O

H

4a,9a-dihydroanthracene-9,10-dione anthracene-9-carboxylic acid 9H-fluoren-9-onediphenylmethanone

Some aromatic carbonyl compounds have a low-lying * excited

state and thus have a reasonable quantum yield (e.g. 0.12 for fluorenone in

ethanol at 77 K and 0.01 at room temperature). However, if a *n state lies

only slightly higher in energy, the fluorescence quantum yield strongly depends

on the polarity of the solvent. In fact, in some solvents, the energy of the

*n state can become lower than that of the * state. When the

polarity and the hydrogen bonding power of the solvent increases, the

*n state shifts to higher energy whereas the *state shifts to lower

energy. Therefore, intense fluorescence can be observed in polar solvents and

weak fluorescence in nonpolar solvents (e.g. xanthone). When an aromatic

molecule has a carboxylic group as a substituent, photophysical effects due to

conformational changes can be observed. For instance, anthracene-9-carboxylic

acid exhibits a broad fluorescence spectrum deprived of apparent vibronic

bands, in contrast to its absorption spectrum and to both absorption and

fluorescence spectra of the conjugate base. Such a difference between the

fluorescence spectra of the acidic and basic forms can be explained in terms of


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conformation of the carboxylate group a -COO-, which should be almost

perpendicular to the ring so that the p system of the anthracene ring is only

slightly perturbed. On the contrary, the carboxylic group a -COOH may be in a

position close to the coplanarity of the ring; the resulting interaction induces an

intramolecular charge-transfer character to the *transition. Charge-

transfer fluorescence bands are indeed usually broad and structureless.

However, because the absorption spectrum of the acidic form exhibits vibronic

bands, the rotation of the -COOH is likely to be photoinduced. In general, the

fluorescence of aromatic hydrocarbons possessing a -NO2 substituent is not

detectable. The existence of a low-lying *n transition explains the

efficient intersystem crossing process (e.g. for 2-nitronaphthalene, the quantum

yield for intersystem crossing is 0.83 in benzene solvent at room temperature).

Many nitroaromatics are indeed phosphorescent. However, in some cases, the

quantum yield for intersystem crossing is significantly less than 1. Therefore,

the absence of detectable fluorescence is likely to be due to a high rate of

01 SS internal conversion, which may be related to the considerable charge-

transfer character of the excited state, as a result of the strong electron-

withdrawing power of the -NO2 group. It should be mentioned that many

nitroaromatics undergo photodegradation. For instance, 9-nitroanthracene is

transformed into anthraquinone upon illumination.

1.5.b. Electron-donating substituents: ( COH, COR, CNH2, CNHR,

CNR2)

In general, substitution with electron-donating groups induces an

increase in the molar absorption coefficient and a shift in both absorption and

fluorescence spectra. Moreover, these spectra are broad and often structureless

compared to the parent aromatic hydrocarbons (e.g. 1- and 2-naphthol

compared to naphthalene). The presence of lone pairs of electrons on the

oxygen and nitrogen atoms does not change the *nature of the

transitions of the parent molecule. These lone pairs are indeed involved directly

in p bonding with the aromatic system, in contrast to the lone pairs of electrons


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of carbonyl substituents or heterocyclic nitrogen. To make the distinction

between the two types of lone pairs, Kasha and Rawls suggested using the term

“l orbital” for the lone pair orbital of aromatic amines and phenols. A

significant intramolecular charge transfer character of the relevant transitions is

expected for planar aromatic amines and phenol; this is confirmed by the fact

that the fluorescence spectra are broad and structureless. If, for steric reasons,

the -NH2 group is twisted out of the plane of the aromatic ring, the degree of

conjugation of the “l orbital” is decreased, but the transitions corresponding to

the promotion of an electron from an “l orbital to a p orbital” are still different

(in particular more intense) to the *n transitions involving the lone pairs

of a carbonyl or nitro group. Departure from coplanarity with the aromatic ring

is also pronounced with -OR substituents, whereas an -OH group is nearly

coplanar [13].

1.5.1 Fluorescence Intensity:

The height of emission peak at maximum emission wavelength gives,

intensity of fluorescence (F). The intensity of fluorescent light is directly

related with the concentration of fluorescent solute in solution as given by

following relation.

F = (I0 – I) f --------------------- (1)

If f is constant in the measured excitation range, then the shape of the

fluorescence excitation spectrum is determined solely by the extinction

coefficient of the molecule [14].

1.5.2 Factors affecting on Fluorescence Intensity:

1.5.3 Concentration of fluorescent solution:

The intensity of fluorescence is proportional to the concentration of the

fluorescent compound only in highly dilute solutions and therefore the

concentration of the compound to be assayed is a very important consideration


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in quantitative work [15]. Fluorophor exists as an isolated molecule when

solution is dilute and emission is monomer fluorescence. However, increase the

concentration results into aggregation called excimer or photodimer which

automatically quenches monomer fluorescence. The complexation of

fluorophor with solvent or impurity leads into fluorescence quenching.

Increase in concentration of fluorescent solutions usually results in quenching

at high concentrations and this is often accompanied by wavelength shifts.

Concentration effects can be ascribed to various phenomena including

reabsorption of emitted light, “true” concentration quenching, dimerization or

aggregation, and miscellaneous experimental and instrumental factors. The

examination of concentration effects is of importance because of the

information obtained in this manner about solute-solute and solute-solvent

interactions. Furthermore, the effects of concentration have to be taken into

account in the measurement of quantum yields of fluorescence and relative

fluorescence intensities [16].

1.5.4 Solvent effect:

Fluorescence intensity and wavelength are affected by change of

solvent. For solvent effect it will be convenient to consider three aspects.

a) Purity of the Solvent:

Since fluorescence is highly sensitive technique, it is important that the

solvents used, should themselves be non-fluorescent and free from fluorescent

impurities. These solvents may be used either for extracting the desired

material or for the actual fluorescence measurement. All solvents should be

checked that they do not contain any undesirable fluorescence and otherwise

vigorously purified. Purification of solvents must be carried out before its use

in fluorescence study [17]. The attention should be paid to the cleaning agents

used to clean glassware. The chromic acid absorbs ultraviolet light and it is

preferable to clean cuvettes in nitric acid rather than in chromic acid. Some


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solvents also absorb specific wavelengths of light and therefore care to be taken

by avoiding the fluorescence in the region of their absorption.

b) Aqueous buffer solutions:

The constituents of the buffer also affect the fluorescence. Commercial

buffer solutions contain constituents, which are not always disclosed by the

manufactures [14]. These constituents could have a fluorescence of their own

or they could be quenching agents. In the case of phosphate buffers, an increase

of phosphate concentration frequently leads to a decrease in fluorescence

intensity.

c) Non-aqueous Solvents:

Fluorescence often varies with solvent and several reasons have been

put forth to explain this alternation in fluorescence [18], on the basis of

dielectric constants of the solvent, the association of solvent and solute by

hydrogen bonding , quenching by solvent molecules and ionization. Report on

Indole indicates the same maximum excitation of 285 nm in solvents, but the

wavelength of maximum fluorescence is 297 nm in cyclohexane, 305 nm in

benzene, 310 nm in water. The fluorescence wavelength thus increases with the

dielectric constant of the solvent due to an effect on the -electrons.

1.5.5 Effect of pH:

The effect of pH upon the fluorescence of a compound is of

considerable importance and knowledge of fluorescence brought about by pH

changes in the medium can be valuable from several aspects. The basicity or

acidity of molecules is determined by its electronic structure and this may

undergo detailed changes during excitation from the ground state by the

absorption of light due to changes in the basicity or acidity of a compound

during excitation. A difference in the basicity or acidity of the ground and

excited states will be reflected in differences between the absorption and

fluorescence spectra with change in pH. The nature of the changes will depend


13

on whether the basicity is increased or decreased during ionization. Thus a

compound may be fluorescent only over a short range of pH, as in the case of

sulphapypridine, which is practically non-fluorescent above pH 4 and is

maximally fluorescent at pH 0.5 to 1.0 [19]. Again a compound may be

fluorescent over a considerable range of pH, but over a certain section of that

range it may be much more fluorescent than over the rest, similarly mono

methyl aniline which has a replaceable hydrogen atom behaves exactly like

aniline but dimethylaniline which does not have such a hydrogen, does not lose

it‟s fluorescence at high pH values [20].

1.6 Experimental Parameters:

The experimental parameters generally used to measure the properties of

any luminescent system are,

1.6.1 Absorption spectra

1.6.2 Emission spectra

1.6.3 Excitation spectra

1.6.4 Fluorescence quantum yields

1.6.5 Fluorescence life time

1.6.1 Absorption spectra:

Einstein relation gives the quantization condition for the absorption or

emission of light by an atom or by molecule

h = hc/ = E2 –E1 --------------- (2)

where, E2 and E1 are the electronic energy levels, h is Planck‟s constant and ,

and c are the frequency, wavelength and velocity of the incident photon.

The Beer-Lambert‟s law governs the absorption of energy by a

molecule. According to this relationship

Log10 (I0/I) = c l ----------------- (3)

Where

I0 = intensity of incident light


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I = intensity of transmitted light

= molar extinction coefficient

c = concentration of the solution

l = path length of the absorbing system through which light passes and

Log10 (I0/I) = optical density or absorbance of the material

In practice, the absorption spectrum is plotted in terms of molar

extinction coefficient ( ) against frequency or wavelength.

Figure 1.4 is a typical absorption spectrum for a S0 S1 transition. The

probability of the absorption depends upon the degree of overlap of the wave

function of the lowest vibrational level of the ground state S00 and the wave

function of the vibrational levels of the first excited singlet state S10 S1n.

Fig.1.4: Illustration of the vibrational bands in the absorption and

fluorescence spectra of aromatic hydrocarbons


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The positions of the absorption peaks and its nature are of significance

in the spectroscopic studies. The nature of the absorption spectrum indicates

the monomeric or dimeric state of the molecular systems, which is of

importance to explore whether the ground state dimer or a monomer is excited

in the absorption process [21]. In solution the broad absorption band is an

indication of dimeric nature of molecules in the ground state while the

structured spectrum indicates the existence of monomolecular species [22]. The

absorption of solids is not as structured as in solution. The nature of absorption

bands also gives an idea about the lattice structures of molecular systems under

study and suggests the possibility of formation of dimeric species [8].

Extensive work has been carried out to establish exact relationship

between absorption and emission properties of organic crystalline materials

with a view to explore electronic and molecular structure of materials in

condensed state [23]. The position of the absorption bands in the spectra of

aromatic hydrocarbons depends upon the number of benzene rings and the way

by which they are condensed. In linearly condensed molecules, band of

absorption spectrum moves towards longer wavelength of the electromagnetic

spectrum. This effect is mainly due to decrease in energy difference between

lower excited and ground state of the molecule. As the number of condensed

rings increases this energy difference becomes progressively less in the linear

polynuclear aromatic hydrocarbons [24]. But in non-linear hydrocarbons the

situation becomes reverse and the energy difference increases as the number of

bends (angular condensation) in the molecule increases.

1.6.2. Emission spectra:

Emission spectrum is the plot of intensity of fluorescence vs.

wavelength. The fluorescence emission spectrum is obtained by irradiating the

sample by a wavelength of maximum absorption as indicated by absorption

spectrum of the sample. Figure 1.5 reveals the levels of the ground S0 and

excited state S1 associated with the absorption and emission spectra. It is

observed that the absorption spectra gives data about the vibrational levels of


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the excited state and the emission spectra yields data about the vibrational

levels of the ground state. In most of the organic materials, the emission

spectrum is the mirror image of the absorption spectrum. This relationship is an

indication of the similarity of the respective vibrational wave functions in the

excited and ground states. Birks and Dyson (1963) [25] reported that this

mirror image relationship is not observed in some molecular systems. In such

systems there is some degree of hindrance to the normal relaxation processes in

the excited or ground state.

1.6.3 Excitation spectra:

Excitation spectroscopy of the fluorescent samples gives a further way

of gaining information about the excited states of organic molecules. An

excitation spectrum is obtained by studying variation in the fluorescence

quantum intensity as a function of excitation wavelength. For most of the

organic molecules the quantum yield is independent of excitation wavelength,

this is due to very efficient process of internal conversion from higher excited

states to the lowest excited singlet states. Even for those molecules for which

quantum intensity is invariant with excitation wavelength, the excitation

spectra provide a powerful tool for measuring the absorption spectra of

molecules, which are at too low concentration for detection by absorption

spectrometry.

The excitation spectrum will be identical to the absorption spectrum

where cd 1. The measurement of quantum intensity is limited by the

sensitivity of the spectrofluorometer and that depends upon the intensity of the

excitation source. Parker (1968) [24] estimated that concentrations as low as

10-12

mol dm-3

can be detected by excitation spectroscopy compared with a

minimum concentration of 10-8

mol dm-3

by absorption spectroscopy.

Excitation spectroscopy is also used to determine the quantum efficiency of

energy transfer between donor and acceptor molecules.


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1.6.4 Fluorescence Quantum Yields:

Fluorescence quantum yield is the major characteristics of a

photochemical reaction. The fluorescence efficiency or the quantum yield ( f)

of a fluorophores is defined as the fraction of the incident radiation that is

reemitted as fluorescence. A high quantum yield value near to 1 (100%

efficiency of the emission process) is generally observed with molecules

having large planar conjugated systems that are relatively rigid [11, 26]. More

flexible molecules are more likely to have values of f, while molecules whose

lowest excited state is achieved by n * transition or that contain heavy

atoms such as bromine or iodine are usually non fluorescent. Quantum yield is

characteristic for each fluorescent compound and is independent of the

excitation and emission wavelength.

Determination of relative quantum efficiency at room temperature of the

two substances is practically simple [13]. The measurement with modern light

sensitive spectrofluorometers involves negligible errors due to excessive

absorption of incident light or due to self-absorption. Under such conditions the

rate of fluorescence emission is proportional to the product I0 cl f.

The integrated area under the corrected fluorescence spectrum is

proportional to the rate of emission of fluorescence and thus, if the

fluorescence emission spectra of two solutions or systems are measured with

the same experimental conditions like same instrumental geometry and at the

same intensity of exciting light then the ratio of the two fluorescence intensities

is given by,

)(10

20

1

2

area

area

clI

lcI

F

F

11

22

)(

)(

densityoptical

densityoptical ---------------------- (4)


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1.6.5 Fluorescence life time:

When an electron is promoted to the higher energy state by absorption

of one quantum of radiation, the question is that to how long it will remain in

the excited state before returning to the ground state. The time spent by an

electronically excited molecule in the higher energy state is known as the

natural radiative life time of the atom or molecule. After this time, the system

reverts spontaneously to its original state by emission of radiation of an

appropriate energy. The fluorescence lifetime of most organic molecules is in

the nanosecond region. The fluorescence life time refers to the mean lifetime of

the excited state i.e. the probability of finding a given molecule that has been

excited still in the excited state after time t is e-t/τ

. The general equation relating

the fluorescence intensity I and the lifetime τ is

I = I0 e-t /τ

where I is the fluorescence intensity at time t, I0 is the maximum fluorescence

intensity during excitation, t is the time after removal of the excitating radiation

and τ is the average lifetime of the excited state.

The precise measurement of the observed lifetime is important since it can

be used to calculate the natural lifetime τo or the absolute quantum efficiency,

Фo, if one or the other is known.

τ = Фo τo

The life time measured mostly by Time Resolved Fluorescence Spectroscopy

method.

1.7. Fluorescence Quenching Phenomenon:

The phenomenon of decrease in intensity of fluorescent compound at its

max is known as fluorescence quenching. When one compound diminishes or

abolishes the fluorescence of another, it is said to quench the fluorescence. The

quenching of fluorescence can be brought about in many ways like,

1.7.1 Inner filter effect

1.7.2 Chemical change

1.7.3 Energy degradation


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1.7.4 Energy transfer

1.7.5 Electron transfer

1.7.1 Quenching by inner filter effects:

Inner filter effects are instrumental effects. They have no influence on

the primary process of emission from molecule originally excited but simply

reduce the observed intensity of luminescence within the material being tested

[11, 27- 28]. The two kinds of inner filter effects are,

a) Excessive absorption of the exciting light,

b) Absorption of the luminescence emitted (solute quenching).

Therefore, the fluorescence intensity of most dyes increases linearly

with increasing concentration at relatively low dye concentrations. The

measured intensity is usually proportional to the optical density (OD) of the

dye solution. The fluorescence intensities can be approximately corrected for

the inner filter effects as follows,

]2/)log[( emexabscorr ODODAFF ------------ (5)

Many fluorescent dyes form dimer or higher aggregates in solutions of

higher concentrations particularly in non-polar solvents [29-30]. The second

type of inner filter effect is produced by the absorption of the fluorescence light

either by an excessive concentration of the fluorescent dye itself (self

absorption) or by other solutes. Self-absorption mainly affects the lower

wavelength side of the fluorescence emission band because in this region it

overlaps with the first absorption band. Self-absorption reduces the

fluorescence intensity. The presence of a second solute that absorbs strongly in

the region where the first solute fluoresces will also cause distortion in the

emission spectrum of the fluorescing dye.

1.7.2 Quenching by chemical change:

If a fluorescent compound undergoes a chemical change as a result of

presence of a second compound it could be converted into a non-fluorescent

product. Although, the fluorescence of the compound is quenched by the


20

addition of the second compound, this form of quenching is to be distinguished

from „true‟ quenching during which no net chemical change occur. Quenching

by chemical change can occur in many ways but the end product is usually a

non-fluorescent compound.

Quenching can occur as result of pH changes in the solution. In case of

aniline when acid is added to it, the fluorescence begins to be quenched at pH-6

and at pH-2 the solution is non-fluorescent [11, 31]. This is due to the

conversion of aniline into the non-fluorescent anilinium ion, which is different

chemical species from aniline.

C6H5NH2 C6H5N+H3

Aniline (Fluorescent) Anilinium ion (Non-fluorescent ion)

1.7.3 Quenching by energy degradation:

Such quenching process involves concentration of singlet excited state

molecule into triplet state by energy or electron transfers, so the fluorophores

no longer emits its energy as fluorescence. The quenching affects of oxygen on

certain iodo, bromo and nitro compounds probably may be due to energy

transfer. The quenching of fluorescent molecules by electron donating I-, Br

-,

SCN , and S2O3

and electron accepting anions such as IO3 , NO3 and S4O6

are cases of involving electron transfer between donor-acceptor. In certain

cases energy transfer between the triplet energy level of quencher and excited

singlet level of fluorophore takes place when triplet energy level of quencher

lie below the excited singlet level of fluorophor which is probable in condensed

system, where donor-acceptor are more closer than in solution [32-33].

,

1.7.4 Quenching by energy transfer:

The system of donor – acceptor pair is excited by proper radiation, to

achieve selective excitation of donor molecules. The donor-acceptor pairs are

such that they satisfy the conditions required for efficient transfer of energy.

The acceptor molecule accepts the energy from donor and donor is deactivated


21

to its ground state and acceptor is raised to singlet excited level. The excited

acceptors so formed de-excite to ground level either radiatively or non-

radiatively. In radiative process the fluorescence characteristics of acceptor

molecule is observed. The systematic variation of acceptor concentration

gradually quenches the fluorescence of donor by ET with simultaneous

sensitization of acceptor fluorescence. Förster (Fluorescence) Resonance

Energy Transfer (FRET) is a physical phenomenon described over 50 years

ago, that is being used more and more in biomedical research and drug

discovery today. It describes a non radiative energy transfer mechanism

between two chromophores. FRET relies on the proximity/ distance dependent

transfer of energy from a donor molecule to an acceptor molecule. This energy

transfer mechanism termed Förster resonance energy transfer named after the

German scientist Theodor Förster. The efficiency of FRET depends on the

following basic parameters-

a) the distance between donor and acceptor molecules,

b) the extent of overlap of the emission spectrum of the donor with the

absorption spectrum of the acceptor,

c) the relative orientation of the donor and acceptor transition dipoles,

d) the quantum yield of the donor.

FRET is the radiationless transmission of energy from a donor molecule, which

initially absorbs the energy, to acceptor molecule. The transfer of energy leads

to reduction in donor‟s fluorescence intensity and excited state lifetime and

increase in the acceptor‟s emission intensity. A pair of molecules that interacts

in such manner that FRETS occurs is referred as donor-acceptor pair. As

mentioned above, there should be proximity between donor and acceptor (10-

100 Å) and absorption/ excitation of acceptor must overlap with emission

spectrum of the donor as shown in Fig.1.5. The research group of our

fluorescence spectroscopy laboratory has also contributed on FRET and

analytical relations are established successfully to develop methods for

quantitative estimations of biomolecules such as vitamin B2 and norfloxacin

[35-36].


22

Fig.1.5: Spectral overlap of excitation of acceptor with emission of donor.

1.7.5 Fluorescence quenching by electron transfer:

D+ + A D + A + Hole transfer

D - + A D + A- Electron transfer

D * + A D + A* Energy transfer

Scheme-I: Quenching mechanisms in energy transfer and electron

transfer processes

The quenching resulting from collisional encounters between the

fluorophore and quencher is called collisional or dynamic quenching. In this

type, the quencher must diffuse to the fluorophor during the life time of the

excited state. Upon contact, the fluorophore returns to the ground state, without

emission of a photon. This is a time dependent process. One experimentally

useful type of quenching is due to collisions between quenching agents and


23

fluorophores, and is called collisional or dynamic quenching. A second type of

quenching is static quenching, in which the quenching agent forms a non-

fluorescent complex with the quenching agent. A final type of quenching,

discussed below, is resonance energy transfer. Static and dynamic quenching

requires direct contact between the fluorophore and the quencher. For dynamic

quenching, the result of this contact is loss of the fluorescence pathway for

return to the ground state, although the mechanism can vary significantly with

the quenchers. Some quenchers act by inducing intersystem crossing (oxygen

and iodide are thought to quench by this method). Others, such as aromatic

amines, appear to donate electrons to the excited state. In the case of dynamic

quenching, contact must occur while the fluorophore is in excited state.

Dynamic quenching is exhibits a concentration-dependence that is described by

the Stern-Volmer equation [37],

][11 0

0 QKQkF

FDq ----------------- (6)

where τ0 is the lifetime of the fluorescent state in the absence of the quenching

agent. If the quenching is not known to be due to dynamic quenching, KD is

replaced by KSV.

For dynamic quenching,

00

F

F ----------------- (7)

because the quenching agent decreases the lifetime of the excited state.

Dynamic quenching increases with temperature, because temperature increases

diffusion rates. The term kq is the second order rate constant that describes the

quenching process. It is proportional to the effectiveness of the quencher and

the accessibility of the fluorophore to collisions with the quencher. The

quenching rate constant is actually comprised of two terms,

f0= fQ k0

where fQ is the fraction of collisions that result in quenching, and k0 is the

diffusion controlled bimolecular rate constant which is given by,


24

))((

1000

43

0

0 qfqf DDrr

L

cm

Nk -------------- (8)

where N0 is Avogadro‟s number, rf and rq are the radii of the fluorophore and

quencher, and Df and Dq are the diffusion coefficients of the fluorophore and

quencher. Typical values of k0 for free fluorophores and free quenchers are

~1010

mol-1

dm3sec

-1. If the fluorophore is bound to the surface of a protein, the

k0 will be roughly half of this value due to available surface occupied by the

protein. If the fluorophore is buried within the protein, the value of k0 will be

even smaller, depending on the accessibility of the fluorophore. Static

quenching is the result of the formation of a non-fluorescent complex between

the fluorophore and the quencher. The association constant for the quencher

fluorophore complex describes the effectiveness of a static quencher:

]][[

][

QF

FQK s ---------------------- (9)

where [FQ] is the complex concentration, [F] and [Q] are the concentrations of

free fluorophore and free quencher. The total fluorophore concentration F0, is

given by

[F]0 = [F] + [FQ] ------------------ (10)

]][[

][]0[

QF

FFK s

By rearranging the above equation,

][

1

]][[

][ 0

QQF

FK s

If it is believed that the all of the decrease in observed fluorescence is due to

complex formation then the equation becomes:

][10 QKF

Fs -------------------- (11)

The above equation is identical to the Stern-Volmer equation for dynamic

quenching. Static and dynamic quenching can be distinguished by lifetime

measurements because dynamic quenching reduces the apparent fluorescent

lifetime, while static quenching merely reduces the apparent concentration of


25

the fluorophore. Alternatively, temperature effects can be used to distinguish

the two forms of quenching. Diffusion rate and therefore dynamic quenching

rate increases with temperature. In contrast, complex formation strength tends

to be inversely proportional to temperature and therefore static quenching tends

to be higher at lower temperatures. In some cases, the effect of the quencher is

due to a combination of static and dynamic quenching shown in Fig. 1.6. This

result in a modified equation can be written as [38],

20 ][])[(1])[1])([1( QKKQKKQKQKF

Fssds DD

------- (12)

Fig.1.6: Distinction between dynamic and static quenching

At high quencher concentrations, a dynamic quencher will appear to exhibit

combined quenching. This is thought to be due to the fact that, at high

concentrations, a significant amount of the quencher molecules are already in

close proximity to the fluorophore. Assuming that any quencher within a

sphere surrounding the fluorophore will quench the fluorescence, a modified

Stern-Volmer equation can be derived [39],

1000

][

00

])[1(

VNQ

d eQKF

F --------------- (13)

in which V is the volume of the sphere (V is usually slightly larger than the sum

of the quencher and fluorophore radii). In proteins, more than one population of

fluorophore may be present. This is especially true for tryptophan residues,

where some may be readily solvent accessible, and others may be buried.

Stern-Volmer plots for these proteins frequently curve downward, reflecting

the quenching of the accessible fluorophores. Assuming the buried fluorophore

is not quenched, and fluorescence will be,


26

b

a

a FQK

FF 0

0

][1 ------------------- (14)

where Ka is the Stern-Volmer constant for the accessible quencher. In practice,

it is likely that the buried fluorophore will exhibit some quenching also, and

therefore the curve would be expected to be more complex than is described by

this equation. In quenching experiments with proteins, the quenching agent

may interact with the protein in ways that alter the protein structure or that

affect the degree of quenching observed. One method for examining this is the

use of several quenchers with different properties. Differential quenching by

positively and negatively charged quenchers suggests a charged environment.

Small KD values typically reflect the steric hindrance of quencher-fluorophore

collisions. Quenching studies may allow isolation of signals from different

fluorophores. They may also allow characterization of conformational changes

that alter the accessibility of the fluorophores to the quenching agent.

Fluorescence quenching by electron transfer (ET) is a one electron

reaction in which an electron jumps from an occupied (HOMO) orbital of one

reactant to an unoccupied (LUMO) orbital of the other as shown in Scheme II.

Scheme II: Photoinduced electron transfer mechanism between donor and

acceptor molecules


27

Fig.1.7: The potential energy curves for PET system

Excited state intermolecular electron transfer between donor-acceptor pair

brought about by the absorption of UV or vis. photon are known as PET

reactions. Electron transfer reaction involves the crossing of the surface free

energy of the reactant to the product at transition state which is shown in Fig.

1.7. The excited state of sensitizer can be an electron donor or acceptor. In

either case, quenching by ET between uncharged species leads to a radical ion

pair or a charge-transfer complex. Electron transfer and energy transfer by

electron exchange require a close approach for effective orbital overlap [40-

41]. As a result the “range of effectiveness” of these mechanisms is usually

limited to distances of less than 10 A0. In contrast coulombic energy transfer

does not involve orbital overlap and can be effective from collision distances of

less than 10 A0 and up to separation distances as large as 100 A

0 [42].

Spin conversion is normally observed in both electron and energy

transfer i.e. the overall spin of the radical ion pair (electron transfer) and the

spin of the excited state of acceptor (energy transfer) match the excited state of


28

sensitizer. Triplet-triplet energy transfer is forbidden by the dipole-dipole

mechanism and takes place by electron exchange [40]. This pathway can be

used to generate reactive triplet states that normally cannot be formed by direct

excitation [43]. Singlet-singlet energy transfer, which proceeds by spin-allowed

electron exchange and dipole-dipole interactions, has been used to probe the

structure of biological macromolecules [44].

In 1963 Leonhardt and Weller reported the emission of complexes

between perylene and dimethyl aniline [45]. These stoichiometric complexes

were postulated as forming by electron transfer between the excited singlet

state of perylene and the ground state of the amine. The term exciplex was

eventually coined to describe these complexes [46]. Today, an exciplex is

defined as two-component system in which charge and electronic excitation are

shared by the components. Many quenching reactions that proceed by electron

transfer involve exciplexes. This is particularly true for planer organic

molecules. Exciplex formation involving these molecules correlates with their

redox potentials and dipole measurements to confirm their charge-transfer

nature.

The primary electron transfer involves a transfer of an electron between

an excited-state and ground state molecules to generate a charge transfer

species, while secondary electron transfer refers to the electron-transfer

pathways which follows formation of the charge-transfer complex. These may

include reversible electron transfer to give the ground state reactants, ionic

dissociation into free ions, triplet recombination to generate an excited state of

one of the reactants, the formation of other charge transfer intermediates and

stable products. The ET can involve either electron transfer or an electron

exchange.

1.8 Fluorescence quenching pathways:

The dynamics of quenching electron transfer must take into account the

positions and motions of the reactants in a given molecular environment. The

processes in solutions classify the reaction pathways into two types.


29

i) The reactants are the mobile and free to approach to close distances

within the lifetime of the excited partner.

ii) Structural factors keep the reactants separated at a fixed distance

during the lifetime of the excited partner.

1.8.1 Fluorescence quenching via encounter complex:

In a fluid medium, reactant molecules which are mobile and

unrestrained by structure or environment may form an encounter complex prior

to quenching. An encounter complex can be visualized as an intermolecular

resemble of an excited-state and ground-state molecule usually separated by a

small distance (7A0) and surrounded by several shells of solvent molecules

termed as the innermost shell solvent cage [40,47-48]. The structure of

encounter complex is affected by the size, shape and charges of the reactants

and by their interactions with the solvent cage. Excitation of the sensitizer

usually takes place before formation of the encounter complex. The events for

proceeding of formation of the encounter complex can be described by the

random walk hypothesis. According to this motion, molecules diffuse in

solution by a series of one-dimensional random steps [40]. If we apply this

concept to the formation of encounter complexes, we can envisage reactant

partners approaching one another in series of randomized zig-zag steps.

Eventually the molecules collide, separate and undergo further collisions.

A typical collision for an uncharged small organic molecule has duration

of about = 10-9

-10-10

second. Quenching pathways may take place within the

encounter complex depending upon the driving force of these reactions. During

the lifetime of an encounter complex, the reactants undergo structural (nuclear)

and electronic (orbital) changes. The vibrational fluctuations, which determine

the nuclear barrier of the pathway, have frequencies of 1012

s-1

.Thus the lifetime

of a typical encounter complex is usually of sufficient duration for rate-

determing nuclear changes to take place. Nuclear reorganization is a critical

aspect of electron transfer and must be considered when evaluation of the rate

limiting barriers of this quenching pathway is carried out.


30

In quenching by electron transfer, if the transfer of an electron occurs

during the lifetime of the collision complex (i.e. when the reactant partners are

in contact), the charge-transfer species immediately formed in a “contact ion

pair” (CIP). The collision complex can also separate slightly, undergo electron

transfer and generate a “solvent-separated” ion pair (SSIP). Following electron

transfer solvent molecules rapidly stabilize contact ion pairs and solvent-

separated ion pairs. Thus, a contact ion pair may be “pried apart” by a solvent

molecule and be converted into a solvent-separated ion pair or vice-versa.

Contact ion pairs and solvent separated ion pairs are sometime described as

“germinate” ion pairs provided that each ionic partner is descendant of the

same parental pair. If solvent-separated ions dissociate into the bulk of the fluid

medium where they become separated by a large distance, they are classified as

free and solvated ions. These ionic species are completely independent of one

another analogous to stable free radical and can exist as distinct, randomized,

solvated and long-lived species.

1.8.2 Fluorescence quenching via exciplex:

Fluorescence quenching via exciplex is a particularly important pathway

when the reactants are planar organic molecules capable of forming

“sandwiched” complexes. If the interaction between the reactants, one of which

is electronically excited, is strong, and encounter complex can rapidly form an

intermediate which may have a sufficiently long life time to undergo light

emission. Such intermediates are termed exciplexes and are characterized by

strong binding energies (5.20 Kcal mol-1

), partial charge character on each

reactant molecules and large dipole moments which reflect the degree of

charge transfer [49-50]. Typically, an exciplex is a long-lived, relatively stable

and structured electronically excited species. Experimental support for

exciplexes is provided by the appearance of broad, structure less and long lived

emission, although absorption and chemical reactions may also be used for

characterization.


31

A molecular orbital wave function representation is used to express the

electronic interaction in an exciplex as a summation of possible states

(wavefuntion) [51].

= C1 (D*A) + C2 (DA*) + C3 (D+ A

-) + C4 (D

- A

+) + C5 (DA)

where the coefficients pertain to the relative contribution of each state. If „A‟ is

exclusively an electron acceptor and if ground-state interactions are neglected,

equation 1.5 simplifies to

C1 (D*A) + C2 (DA*) + C3 (D+ A

-) ---------- (15)

If C3>>C2, the exciplex has pronounced charge-transfer character and will have

a tendency to dissociate into radical ion pairs, especially in polar solvents. In

the extreme case, an exciplex is more accurately described as a contact ion-

pair. Emission from exciplexes proceeds by a vertical, Franck-Condon allowed

transitions from a minimum on an excited state surface to a low-lying ground

state surface involve a significant nuclear change, the emission may not be

observed. Other decay processes, such as ionic dissociation into solvent-

separated radical ions or chemical reactions may prevail. Such a non-emitting

charge-transfer species is described as a “non-emitting” exciplex and has

properties similar to those of a contact ion-pair. For C3>>C2, exciplex

formation leads to energy transfer. In such a case, exciplexes tend to emit light

or undergo separation to form the excited state of the acceptor and ground state

of the donor.

1.9 Surfactants:

Surfactants are characterized by a hydrophilic charged „head‟ and a

hydrophobic hydrocarbon „tail‟. The most outstanding property of surfactants is

their tendency to form aggregates, the micelles, at sharply defined critical

micelle concentration (CMC). The micelles formed by ionic amphiphilic

molecules in aqueous solutions are dynamic association of surfactant molecules

that achieve segregation of their hydrophobic proteins from the solvent via self

assembly [52].


32

The aggregation of single molecules of a certain type when dissolved in

water, to form a particle of colloid dimension called a micelle which is shown

in Fig.1.8. The molecules or monomers, which can take part in these processes,

are characterized by possessing two regions in their chemical structure. One is

hydrocarbon chain, the hydrophobic region of the molecule, and other is an

ionized group or water soluble group, the hydrophilic region of the molecules

e.g. Sodium lauryl sulphate.

Fig.1.8: Structure of micelle

The existence in one compound of two moieties, one of which has affinity for

the solvent and other is antipathetic to it, has been called amphipathy by Hartlet

[52]. This dual nature is responsible for the properties of micellization, surface

activity and solubilization. As a class, these substances, which include soaps

and detergents, can be called associate in solution forming particles of colloidal

dimensions. Due to their tendency to become adsorbed at interfaces, they are

often called surface-active agents or colloidal surfactants.


33

1.9.1 Classification of surfactants:

From the commercial point of view surfactants are often classified

according to their use. The most accepted and scientifically sound classification

of surfactants is based on their dissociation in water.

1.9.2 Anionic surfactants:

Anionic surfactants are dissociated in water in an amphiphilic anion, and

a cation, which is in general an alkaline metal (Na+, K

+) or a quaternary

ammonium ion. They are the most commonly used surfactants. They include

alkylbenzene sulfonates (detergents), (fatty acid) soaps, lauryl sulfate (foaming

agent), di-alkyl sulfosuccinate (wetting agent), lignosulfonates (dispersants)

etc. Anionic surfactants account for about 50 % of the world production.

Sr. No Name Structure

1 Potassium laurate CH3(CH2)10COO- K+

2 Sodium dodecyl (lauryl)

sulphate

3 Hexadecylsulphonic acid CH3(CH2)15SO-3 H

+

4 Sodium

dioctylsulphosuccinate

1.9.3 Cationic surfactants:

Cationic surfactants are dissociated in water into an amphiphilic cation

and an anion, most often of the halogen type. A very large proportion of this

class corresponds to nitrogen compounds such as fatty amine salts and

quaternary ammonium salts with one or several long chain of the alkyl type,

often coming from natural fatty acids. These surfactants are in general more

expensive than anionic surfactants, because of the high-pressure hydrogenation

reaction to be carried out during their synthesis. As a consequence, they are

only used in two cases in which there is no cheaper substitute, i.e. (1) as

CH3(CH2)11SO-4 Na+

C8H17OOCHSO-3 Na+

C8H17OOCCH2


34

bactericide, (2) as positively charged substance which is able to adsorb on

negatively charged substrates to produce antistatic and hydrophobic effect,

often of great commercial importance such as in corrosion inhibition.

Sr.

No

Name of Surfactant Structure

1 Hexadecyl (cetyl)trimethylammonium

bromide

CH3 (CH2) 13N+(CH3) 3 Br

-

2

Dodecylpyridium chloride N

+

C12H25Cl-

3

Dodecyl hydrochloride CH3(CH2)11NH3

+

1.9.4 Nonionic surfactants:

Nonionic surfactants account about 45% of the overall industrial

production. They do not ionize in aqueous solution, because their hydrophilic

group is of a nondissociable type, such as alcohol, phenol, ether, ester, or

amide. A large proportion of these nonionic surfactants are made hydrophilic

by the presence of a polyethylene glycol chain, obtained by the

polycondensation of ethylene oxide and they are called polyethoxylated

nonionics. In the past decade glucoside (sugar based) head groups, have been

introduced in the market, because of their low toxicity. As far as the lipophilic

group is concerned, it is often of the alkyl or alkylbenzene type, the former

coming from fatty acids of natural origin. The polycondensation of propylene

oxide produce a polyether which (in opposition to polyethylene oxide) is

slightly hydrophobic.


35

Sr.

No.

Name of the surfactant Structure

1 Polyoxyethylene p-tertoctylphenyl

ether

C8H17C6H4O(CH2CH2O)10H

2. Polyoxyethylene monohexadecyl

ether

CH3(CH2)15(OCH2CH2)21OH

1.9.5 Zweterionic or Ampholytic surfactant:

This type can behave either as an ionic, non-ionic, or cationic species,

depending on the pH of the solution. When a single surfactant molecule

exhibits both anionic and cationic dissociations it is called amphoteric or

zwitterionic. This is the case of synthetic products like betaines or sulfobetaines

and natural substances such as amino acids and phospholipids.

1.9.6 The micellization process:

The most important property of amphipathic molecules or ions

characterized by a polar hydrophilic head and non-polar hydrocarbon tail is

their tendency to form large aggregates, the micelles, above a certain rather

sharply defined concentration. So micelle is defined as “a colloidal particle

together with its surrounding stabilizing agent” [50]. Surfactant molecules (e.g.

CTAB, SDS, Triton X-100, Brij – 35 etc.) self-aggregate into supramolecular

structure when dissolved in water or oil. The simplest aggregate of these

surfactant molecules is called a micelle, and dispersion of the aggregates in

water or oil is referred to as micellar solution [53-55]. A typical micelle has

size of 50 Å and is made of about 100 surfactant molecules [56]. The self-

association gives rise to rich variety of phase structure shown in Figure1.9.

Aggregation is not only limited to aqueous solution but also it is some time

observed in non-aqueous polar solvents such as ethylene glycol and non-polar

solvents such as hexane [57].


36

1.9.7 Solubilization of organic compounds:

Self-association property of surfactants to form self-assembled

aggregates, the micelles, in an aqueous medium has a profound effect on the

solubility of the some organic substances (additives) which are otherwise

sparingly soluble in water [58-59]. Solubilization by micelles is of importance

in many industrial processes such as detergency, emulsion polymerization, oil

recovery, etc. and in a variety of fundamental research oriented polymerization

like micellar modeling of biological membranes [60].

The solubilization can be ascribed to incorporation of hydrophobic

substance in to micelle in solution of surfactant. Solubilization has been treated

as partitioning of additive molecules (solubilizates) between a micellar phase

and intermicellar bulk phase [61-63]. The partitioning behavior of

solubilization between intermicelle bulk phase and micellar phase is an

indication of the hydrophilic – lyophilic balance of the molecule. It has been

discussed that the affinity of water for the solubilizates is important in

partitioning [64] due to water dragging effect where the water is carried as a

shell around the solubilizate. The partition coefficient is dependent on the

structure of solubilizate and the surfactant that constitutes the micelles.

In addition to the solubilization equilibrium, the micro viscosity of the

micellar interior and location of the solubilizate within the micelles is also

important in many applications of micellar solubilization. The physical

behavior of the surfactant micelles can be visualized as the construction of

model membrane to biological surface with the solubilizate, drug carrier and

drug release [65-66].

1.9.8 Mechanism of solubilization:

Solubilization is believed to occur at five sites in the micelle as shown in

Scheme-III.

1. the surface of the micelle at the micelle solvents interface

2. between the hydrophilic head group


37

3. palisade layer of the micelle between the hydrophilic ground and the

first few carbon atoms of the hydrophobic groups that comprise the

outer core of the micellar interior.

4. more deeply in the palisade layer

5. in the inner core of the micelle

Ionic micelles ordinarily have an extensive hydrophobic core region,

which can interact strongly with hydrocarbon and halogenated hydrocarbons of

solutes. Hydrophobic effects have often been considered to be dominating in

determining the locus of solubilization [67-68]. Surfactant micelles can be

pictured as having a highly nonpolar interior and a relatively polar interfacial

region. Therefore nonpolarized or easily polarizable compounds are solubilized

in aqueous medium in the inner core of the micelle between the ends of the

hydrophobic groups of the surfactant molecules. Polarized hydrocarbons are

solubilized by absorption at the micelle-water interface and by replacing water

molecules that may penetrate the core of the micelle close to the polar heads,

but solubilization of additional material is deep in the palisade layer as located

in the inner core of micelle [59]. The polarizability of the π electron cloud of

the aromatic nucleus and its consequent ability to interact with the positively

charged groups at the micelle water interface may account for the initial

adsorption of these hydrocarbons in that location [69].

Scheme-III


38

1.10 Applications:

1.10.1 PET in synthetic organic chemistry:

The photoinduced electron transfer reactions are of interest to

photochemists to understand the generation of charge transfer intermediates

and stable products when donor and acceptor molecules are placed close in

suitable solvents. Many reactions photosynthesized by electron transfer were

investigated and studied thoroughly to know mechanism and probable

products.

1.10.1.a Reaction of alkanes:

The most clear cut examples of electron transfer phtosensitizers are

those involving electron deficient sensitizers and electron rich olefins. The

radical cations generated from these reactions have been shown to undergo

polymerization, dimerizations, cross cycloaddition, nucleophilic substitution

and isomerization [70-72].

1.10.1.b Bond cleavages, ring opening’s elimination and isomerization:

Strained cyclic organic molecules are efficient quenchers of excited

states. These reactions frequently involve electron transfer. Some examples are

photolysis of methanolic solutions of tricyclo heptanes and 1-cyanonaphthalene

leads to CH3OH addition [73]. Sensitized bond cleavage reactions of 1,2-diaryl

ethanes and aryl pinacols have been reported [74].

1.10.1.c Valence isomerization:

The rearrangement of n-bornadiene to the richer quadricyclane and the

reverse reaction can take place on photolysis in polar solvents in the presence

of electron deficient photosensitizers [75]. Electron transfer from valence

isomer to the excited states of electron deficient senzitizer is supported by

thermodynamic consideration. In the presence of singlet and triplet sensitizer,

the ring opening of quadricyclane displays a significant solvent dependence

[76].


39

1.10.1.d Photo-oxygenations:

Photo-oxygenations have been considered to proceed via energy transfer

quenching, involving sensitization of oxygen by sensitizer triplet to form

excited singlet oxygen, which is reactive intermediate. A typical example

involves the oxygenation of electron rich olefins conjugated to aromatic

moieties with electron deficient sensitizers for example 9,10-dicyanoanthracene

[77]. The search for new photosensitizer would be always of novel applications

to synthetic organic chemistry.

1.10.1.e Photo-reduction of dyes by electron transfer processes:

Photo reduction is observed for some dyes in which the dye appears to

be bleached in light. In such reduction processes two „H‟ atoms (two electrons

and two protons) are added to the dye molecule D.

D + RH2 DH2 + R

The photo-reaction goes against the thermochemical gradient and

reverses spontaneously in the dark. The most important example of such

reactions is the photosynthesis in the plants. The light absorbed by the

chlorophyll molecules promotes an electron energetically to excited state.

Thus, provides the driving force for the primary processes. Such a

photosynthetic process occurring in the plants can be developed in the

laboratories. The operation of artificial photosynthetic devices depends mainly

on photoinduced electron transfer processes (PET) and hence PET reactions are

of immense importance in this context [78].

1.10.1.f Photo-oxidation reactions by electron transfer:

Photo-oxidation reactions in absence of molecular O2 are better

described as photochemical oxidation-reduction reaction in which an electron

transfer will occur between donor and acceptor. Photo redox reactions can be

mediated by an intermediate, which is called as sensitizer acts as photo catalyst

h


40

in more generation sense. Thus, the photoinduced electron transfer reactions

are significantly broadened the scope of organic photochemistry.

1.10.2 PET systems as sensors:

Upon excitation the luminophores, especially those with groups held

outside but close to the -electron systems of the luminophore, show an

increased susceptibility to redox reactions. Knowledge of the redox potential of

their reactions can be used to tailor PET processes that can be switched on or

off. In the off state, the excited state energy must be bigger than that require to

oxidize the receptor and to reduce the fluorophore. Thus, allowing PET from

receptor to fluorophore. In the on state, a cation in the receptor cavity raises to

oxidation potential, causing the thermodynamic condition for PET to be

removed and fluorophores to undergo emission. A sensor molecule when

subjected to light energy, an electron is transferred from the receptor to

fluorophore, taking energy from its excited state and rendering the system non-

luminescent. However, if the receptor traps a guest, its lone pair of electrons

becomes bound up, electron transfer does not occur and the excited state

energy of the system is released as emission from the fluorophore [79].

1.10.3 Molecular Information Processors:

Molecular information processor is an interesting device based on PET

and consists YES, NOT AND or XOR and other logic gates. The use of

chemical input and photonic output is developing field of research. For

example, a YES gate molecule is one that fluoresces in the presence of H+ and

a NOT gate molecule is one whose fluorescent emission disappears when H+

is

added. This is an important area of research and it is now obvious that this type

of work hold out the potential for molecular computers and neutral networks

using photon mode input.


41

1.10.4 Qualitative and quantitative applications of PET:

PET reactions are of significance in the fields of chemistry and biology.

Extensive theoretical and experimental studies in homogeneous media and

organized assemblies have been done for more than a couple of decades for the

better understanding of PET reactions. PET is used for spectroscopic detection

of biomolecules, drugs and nucleic acids and also to understand molecular

interactions between them. The detection methods involved use of fluorescent

probe whose intensity is quenched regularly by photoinduced electron transfer

between ground state quencher to excited fluorescent probe. The gradual

quenching of fluorescence of probe as a function of concentration of quencher

enabled to establish quantitative relation between extent of quenching of

fluorescence and concentration of quencher that is an analyte. The earlier

spectroscopic methods involved separation of an analyte from other

components or impurities and its detection based on Beer‟s law in direct

absorption methods and in direct fluorimetric methods. These methods are

laborious and require that the analyte should be fluorescent. The electron

donor-acceptor pair should be such that donor should be capable to donate

electron i.e. easily oxidisable and other reducible by accepting the electron

from donor.

1.11 Literature survey on PET process:

Photoinduced intermolecular electron transfer interaction between

coumarin dyes and aromatic amines has been investigated in sodium dodecyl

sulphate micellar solutions using steady state and time resolved fluorescence

quenching measurements [80]. In the time-resolved fluorescence

measurements, the analysis of the fluorescence decays following a micellar

quenching kinetics model assuming a unified quenching constants (kq) per

quencher occupancy does not give satisfactory results especially for the higher

quencher concentrations used. The correlation of the observed kq values in the

micellar solutions with the free-energy changes ( G0) for electron transfer


42

reactions show an inversion in the observed rates as predicted by Marcus outer

sphere electron transfer at exergonicities more than 0.65eV are discussed [80].

Photoinduced electron transfer between octadecyl-rhodamine B (a hole donor)

and N, N-dimethyl -l-naphthylamine (hole acceptor), located in the head group

region of sodium dodecyl sulfonate micelles, has been examined for different

acceptor concentrations using time resolved fluorescence. The experimental

results were analysed using the Marcus distance-dependent transfer rate

modified to take into account the heterogeneous nature of micelles. Diffusion

of the donor or acceptor is included in the theoretical analysis and the results

are compared with earlier experiments involving the same donor-acceptor

combination but in a different type of micelles [81].

Torimura et al. studied the fluorescence quenching by photoinduced

electron transfer between various fluorescent dyes and nucleobases. In these

systems nucleobases are used as electron donors. Appropriate donor-acceptor

pair was selected from electrochemical data obtained by cyclic voltammetry.

The quenching experiments were performed by keeping concentration of

acceptor to some constant value and varying that of donor in aqueous solutions

[82].

S.P.Wu et al. studied the fluorescence quenching study on the

interaction of bases of nucleic acid with electron accepting sensitizer and serum

albumin containing electron rich tryptophan residue. The fluorescence

quenching data shows that nitrogen bases act as effective electron donors to

quench the fluorescence of electron-accepting sensitizers. The quenching by

diffusion-controlled rate coincided well with the static and dynamic Stern-

Volmer correlation. The free energy changes for photo induced electron

transfer process were also investigated [83].

Z.P. Du et al. reported the experimental study on the photo interaction

between sanguinarine and guanosine by using UV-visible and fluorescence

spectroscopy. The binding of sanguinarine to guanosine was substantiated by

the hypochromism and bathochromism in the absorption spectra and the

emission quenching in fluorescence spectra. These spectral features strongly


43

support the interactions of sanguinarine with guanosine. The thermodynamic

data for the sanguinarine binding to guanosine were also calculated .The results

show that the binding of sanguinarine to guanosine is not only exothermic but

entropy driven [84]. Similarly X.Zhang et al. reported a spectral study of the

interaction between chelerythrine chloride and adenosine by UV-visible

spectrophotometric and spectrofluorimetric measurements and calculated the

thermodynamic parameters [85].

B. Chakraborty et al. studied the interaction of proflvin with

triethylamine in homogeneous and micellar media and photoinduced electron

transfer probed by magnetic field effect. The two prime phenomena were

explored in this study are photoinduced electron transfer (PET) and ground –

state complex formation. The experimental result shows that it is the medium,

which determines the reaction pathways to be followed. Magnetic field effect

helps to elucidate the reaction mechanism involved in this system [86].

Neeti Singh et al. reported spectrophotometric study of the charge

transfer complexation of picric acid as an electron acceptor with p-nitroaniline

as an electron donor in three different organic solvents. The results were

discussed in terms of association constant (kct), molar extinction coefficient

( ct), standard free energy ( G0), oscillator strength (f), and transition dipole

moment ( EN). The results reveal that the interaction between the donor and

acceptor is due to - * transitions. The stoichiometry of the complexes was

found to be 1:1 due to linear relationship between donor and acceptor [87].

A. Chakraborty et al. investigated the PET between different coumarin

dyes and N, N-dimethyl aniline in AOT reverse micelle using steady state and

time resolved fluorescence spectroscopy. The fluorescence quenching data

shows that slower electron transfer rate in reverse micelle in comparison to that

in the neat solvent. It was observed that the retardation in the ET rate in the

correlation of the free energy change with the ET rate [88].

F. Yu et al. developed a new spectrofluorimetric method for

determination of adenosine disodium triphosphate (ATP). The interactions

between prulifloxacin (PUFX)-Tb3+

complex and ATP has been studied by


44

using UV-visible absorption and fluorescence spectra using these pairs as

fluorescence probe, under the optimum conditions. ATP can remarkably

enhance the fluorescence intensity of the complex and enhanced fluorescence

intensity is in proportion to the concentration of ATP. The dynamic range and

detection limit of ATP was investigated [89].

1.12 Aim and scope of the present work:

The fluorescence quenching phenomenon first described over 50 years

ago and since then being used more and more in biomedical research. It is well

established that physico-chemical interactions between the components of

nucleic acids such as nitrogen bases, nucleosides, nucleotides and fluorescent

probe resulting into the strong fluorescence quenching of the latter, have been

the focus of extensive recent research. Several mechanisms appear to operate in

such systems namely quenching due to chemical reaction, quenching involving

electronic energy transfer, quenching due to the heavy atom and quenching

processes proceeding via charge transfer interaction. Photoinduced electron

transfer (PET) appears to be the most likely mechanism of quenching since the

redox potentials are of the appropriate magnitudes for polycyclic aromatic

fluorescent probe and nucleic acid couples. This mechanism is of intrinsic

interest because it takes place in aqueous or partially aqueous environment and

thus providing opportunities for studying the effects of hydrophobic

interactions, hydrogen bonding, and proton transfer coupled to PET

fluorescence quenching phenomenon.

The components of nucleic acids have an important function in life

process, so study on them has become an important research field of life

science. Quantitative determination of nucleic acid is required in many fields,

such as molecular biology, biotechnology and medical diagnosis. The natural

fluorescence intensity of nucleic acids is so weak that the direct use of their

fluorescence emission properties is limited. Therefore, to study the components

of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) using the

fluorescence method an extrinsic fluorescent probe should be introduced.


45

Different probes react with nitrogen bases, nucleosides and nucleotides in

different ways, DNA and RNA molecules are known to enhance the

fluorescence intensity and also to quench the fluorescence of some of the

probes such as Ethidium bromide, sanguinarine, chelerythrine chloride,

proflavine, pyrene, coumarine etc. which are reported in literature. DNA and

RNA are composed of the components like nitrogen bases, nucleosides and

nucleotides. Adenine, guanine, uracil, thymine and cytosine are the nitrogen

bases. Adenosine, guanosine, uridine and cytidine are nucleosides while

adenosine 5‟ monophosphate (AMP), guanosine 5‟ monophosphate (GMP) and

uridine 5‟ monophosphate (UMP) are the nucleotides. Many of these

biomolecules constituting nucleic acids are of pharmaceutical importance.

Uracil is used for drug delivery and in the body it helps to carry out the

synthesis of many enzymes necessary for cell functions. Its derivatives 5-fluoro

uracil and 5-methyl uracil are known to be antiviral, anticancer drugs. These

materials are weakly photosensitive and fluoresce too low to detect in direct

methods of analysis. Therefore in present work attempts were made to have

systems in which uracil and its derivatives bind with fluorescent 9-anthracene

carboxylic acid (9-ANCA) probe and form charged pairs in aqueous medium.

Such pairing was expected to form charge transfer state upon excitation and

may results in quenching of fluorescence of probe molecule. The possibility of

binding between these molecules was examined by electrochemical studies of

such pairs which helped further to consider photoinduced electron transfer

(PET) from nitrogen base molecules to the fluorescent probe.

Similarly, AMP, GMP and UMP are the structural units of the nucleic

acids used as drugs. AMP is an intermediary substance formed during the

body‟s process of creating energy in the form of adenosine triphosphate (ATP)

from food. The body creates AMP within cells during normal metabolic

processes. It has medicinal importance and its injection available commercially

is used in post therapeutic neuralgia treatment. In brain research studies UMP

is used as a convenient delivery compound for uridine. The present work aimed

to develop fluorimetric method for analysis of these molecules by using 9-


46

ANCA. The direct molecular interaction between 9-ANCA and nucleotides is

not possible as both being isoelectronic. The cationic surfactant such as cetyl-

trimethyl ammonium bromide (CTAB) is known to solubilize, stabilize the

drugs and DNA, RNA molecules of opposite charges and may result in several

desirable or undesirable interactions. Hence possible electrostatic interactions

between the pairs of 9-ANCA-CTAB and CTAB-nucleotides are used to

develop a new fluorimetric method for analysis of nucleotides from aqueous

solution and applied successfully for pharmaceutical samples containing

nucleotides. The possible electrostatic interactions between these pairs were

examined by conductometric studies. In addition to 9-ANCA, perylene

solubilised in CTAB was found to be suitable probe for detection of these

biomolecules by using PET.

The nucleoside like adenosine used as anti-inflammatory agent also

undertaken in PET studies with proflavine hemi sulphate (PF). The ground

state charge transfer complex formation was observed by absorption and

fluorescence studies.

Studies on photophysical properties of fluorescent organic materials in

aqueous solution were performed. The materials such as 9-ANCA, perylene

and PF were tested for photoabsorption and emission performance with a view

to use as probe and to develop analytical techniques for determination and to

understand the molecular interaction with DNA and RNA molecules. The

spectral data obtained would found useful in deciding the donor (components

of DNA and RNA) and acceptor (fluorescent probe) pairs for PET studies

which are satisfying basic condition required for the electron transfer process.

The PET experiments are planned in aqueous and micellar solutions to examine

optimum conditions. Attempts were made to verify Stern-Volmer relation and

thus to determine the binding parameters to understand molecular interaction

between donor and acceptor. Thus, studies carried on different electron donor –

acceptor pairs would found useful in developing new analytical methods for

detection as well as to understand the molecular interaction based on PET.


47

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