CHAPTER 1-Luminescene Topics

CHAPTER – I Introduction
Chapter-I: Introduction
1.1 Luminescence
Luminescence is the emission of light from any substance and occurs from
electrically excited state. The term “Luminescence” was introduced in to 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 [1, 2]. This definition reflected an important
property, but it did not distinguish luminescence from other type of glow that
are also excess emission over and above thermal emission background, among
which are reflected and refracted light. 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
thermal emission of a body. If this emission has a duration considerably
exceeding period of light oscillations [1-3]. Luminescence is the phenomenon
in which all or part of absorbed energy is re-emitted in the form o
electromagnetic radiation in the visible or near visible region of the spectrum.
The luminescence involves at least two steps, the excitation of the electronic
system of he material and the subsequent emission of photons. An example of
luminescence is the light or glow emitted by a luminous watch dial.
Luminescence contrasts with incandescence which is the production of light by
heated materials [1-5].
When certain material absorbs various kinds of energy, some of the energy
may be emitted as light. This process involves two steps:
1. The incidental energy causes the electron of the atoms of the absorbing
materials to become excited and jump 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 1/100, 00 of a
sec.) or long (many hours). If the interval is short, the process is called
fluorescence, if the interval is long, the process is called phosphorescence. In
1
either case the light produced is almost always of lesser energy and of longer
wavelength than the exciting light [1, 3, 4].
1.1.3. Jablonski Diagram:
Processes which occur between the absorption and emission of light are
usually illustrated by Jablonski diagram. A typically Jablonski diagram is
shown in Figure. 1. The ground, first and second electronic states are depicted
by S0, S1 and S2, respectively. At each electronic energy levels the fluorophores
can exist in a number of vibrational energy level (denoted 0,1,2, etc).
Transitions between states are depicted as vertical lines to illustrate the
instantaneous nature of light absorption. Transitions occur in about 10-15
seconds, a time too short for significant displacement of nuclei. (Frank-Condon
principal) [5]
1.1.1 Phosphorescence:
Phosphorescence is a emission of light from triplet-excited state, in which
the electron in the excited orbital has the same spin orientation as the ground-
state electron. Transition to the ground state are forbidden and the emission rate
are slow (10-3-100S-1), so phosphorescence lifetimes are typically milliseconds
to seconds. Phosphorescence is usually not seen in fluid solutions at room
temperature, but there are many deactivation processes that compete with
emission, such as nonradiative decay and quenching processes. [6]
1.1.2. Fluorescence:
Fluorescence is emission light from singlet-excited states, in which the
electron in the excited orbital is period (of apposite sign) to the second electron
the excited orbital. Return to the ground state is spin-allowed and occurs
rapidly by emission of a photon [4, 7, 8].Fluorescence is usually excited to
some higher vibrational level of either S1 or S2. 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 take 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
2
that the emission spectrum is typically a mirror image of the absorption

spectrum of the S 0 ⎯
⎯→ S1 . This emission rates of fluorescence are typically
108 s-1, so that a typically fluorescence lifetime is near 10ns. 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.
1.2. Types of Fluorescence:
The fluorescence is observed as long as excitation is in process and is
known as steady state or prompt fluorescence. When excitation is cut off,
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
3
1.3. Processes competing with Fluorescence:

1.3.1 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
S1. it is from this position that photon will be emit [Kasha’s Rule]. As a
consequence, fluorescence form solution, when it occurs, always involves a
transition from the lowest vibrational level of an excitation level of an excited
state [4, 9]. A consequence of the efficiency of vibrational relaxation is that he
fluorescence band for a given electronic transition is displaced toward lower
frequencies or longer wavelengths from the absorption bands (the stock effect).
1.3.2 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.
When a molecule is excited to an energy level higher than the lowest
vibrational level of the first electronic state, vibrational relaxation (and internal
conversion if the singlet excited state is higher than S1) leads the excited
molecule towards the 0 vibrational level of the S1 singlet state with a time-scale
of 10-13–10-11 s. [10]
From S1, internal conversion to S0 is 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.
1.3.3 Intersystem Crossing (ISC):
Intersystem crossing is a non-radiative transition between two isoenergetic
vibrational levels belonging to electronic states of different multiplicities. For
4
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; 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 S0→S1)Crossing
between states of different multiplicity is in principle 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 → π * 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 [11]
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 a
significance in producing delayed fluorescence and phosphorescence which is a
radiative decay of triplet state molecule to the ground state, are summarized in
Fig. 1.2. .
Fig.1.2. De-Excitation pathway of excited Molecules
5
1.4. Experimental Observables:

Following experimental observables are used to measure the properties
of any luminescent system.
1 Absorption spectra
2 Emission spectra
3 Excitation spectra
4 Quantum efficiency and yields
1.4.1 Absorption spectra:
The quantization condition for the absorption or emission of light by an
atom or by molecule is given by Einstein relation
hν = hc/λ = E2 –E1 --------------- (1)
where, E2 and E1 are the electronic energy levels, h is Planck’s constant and ν,
λ and c are the frequency, wavelength and velocity of the incident photon.
The absorption of energy by a molecule is governed by the Beer-
Lambert’s law. According to this relationship
Log10 (I0/I) = ε c l ----------------- (2)
Where
I0 = intensity of incident light
I = intensity of transmitted light
ε = molecular 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 molecular
extinction coefficient (ε) against frequency or wavelength.
Figure 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.
6
Fig. 1.3. Illustration of the vibrational bands in the absorption and

fluorescence spectra of aromatic hydrocarbons.
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 [12]. 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[13]. But
in solids the absorption spectra are 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 [14].
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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 [15-18].
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, longest wavelength 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[19]. But in
non-linear hydrocarbons the situation becomes reverse. The energy difference
in general increases as the number of bends (angular condensation) in the
molecule increases.
1.4.2. Emission Spectra:
The molecule is in the lowest vibrational level of the ground state at
room temperature and on absorption of visible or ultraviolet radiation the
molecule gets excited to the higher electronic energy level. This transition
occurs too rapidly within ~ 10-15 sec. The molecule now has the possibility to
drop down to the ground state with the emission of light. The fluorescence
always takes place from the lowest vibrational level of the first excited singlet
state and the shape of the fluorescence emission spectrum is independent of the
wavelength of the exciting light.
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 3. 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
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
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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) [20] 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.
1.4.3. Excitation spectra:
Excitation spectroscopy of the fluorescent samples gives a further way
of gaining information about the excited states of organic molecules shown in
Fig.3. An excitation spectrum is obtained by examining variation in the
luminescence quantum intensity as a function of excitation wavelength. For
most of the organic molecules the quantum yield is independent of excitation
wavelength because of 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, excitation spectra provide a powerful tool for measuring
the absorption spectra of molecules, which are at too low a concentration for
detection by absorption spectrometry.
The excitation spectrum will be identical to the absorption spectrum

where εcd<<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) [21] estimated that concentrations as low as
10-12 M can be detected by excitation spectroscopy compared with a minimum
concentration of 10-8 M 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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1.4.4 Fluorescence Quantum Yields:
Of the excited molecules into the higher excited states all will not return
to the ground state by fluorescence or phosphorescence process because these
processes competed by IC and ISC.
The quantum efficiency of fluorescence Φf is defined as the ratio of the

number of fluorescence photons emitted to the number of photons absorbed. It
is important to distinguish at same stage the difference between the term’s
quantum efficiency and quantum yield. The term quantum yield is used to
define the fraction of excited molecules, which may follow a given pathway
either radiatively or non-radiatively. The methods of determination of absolute
quantum efficiency need to compare the rate of absorption of excitation light
with the total rate of emission of fluorescence at all wavelengths and in all
direction. In principle this is simple but in practice it is a difficult experiment to
perform with precision.
The most direct method for determination of fluorescence efficiency

was reported by Vavilov [22], Waber and Teale [23]. They have devised an
absolute method in which a standard fluorescent solution is not required and in
addition to this the method is applicable to dilute solutions also. Determination
of relative quantum efficiency at room temperature of the two substances is
practically simple [24]. 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 ε c l Φf.
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/ sytems are measured with the
same experimental conditions like same instrumental geometry and at the same
intensity of exciting light. The ratio of the two fluorescence intensities is given
by
10
F2 I 0 ε c l Φ 2 (area )
= =
F1 I 0 ε cl Φ 1 (area)
Φ 2 Χ ( optical density ) 2
=
Φ 1 Χ ( optical density ) 1
1.5. Structural factors on Fluorescence Measurments:
1.5.1. 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 exhibit low fluorescence quantum yields, as explained
above. The dominant de-excitation pathway is intersystem crossing (whose
efficiency has been found to be close to 1 for benzophenone).
O O
O O
O
O
diphenylmethanone 4a,9a-dihydroanthracene-9,10-dione anthracene-9-carboxylic acid 9H-fluoren-9-one
Some aromatic carbonyl compounds have a low-lying π → π * 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 → π * state lies
only slightly higher in energy, the fluorescence quantum yield strongly depends
on the polarity of the solvent (proximity effect). In fact, in some solvents, the
energy of the n → π * state can become lower than that of the π → π * state.
When the polarity and the hydrogen bonding power of the solvent increases,
the n → π * state shifts to higher energy whereas the π → π * 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
11
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 (similar to the anthracene spectrum).
Such a difference between the fluorescence spectra of the acidic and basic
forms can be explained in terms of 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
π → π * 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 an -NO2 substituent is not detectable. The existence of a low-lying
n → π * transition explains the efficient intersystem crossing process (e.g. for
2-nitronaphthalene, the quantum yield for intersystem crossing is 0.83 in
benzene solution 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 S1 → S 0 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.
1.5.2. 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
12
compared to the parent aromatic hydrocarbons (e.g. 1- and 2-naphthol

compared to aphthalene). The presence of lone pairs of electrons on the oxygen
and nitrogen atoms does not change the π → π * 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 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
orbitals 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 → π * transitions involving the lone pairs of a carbonyl or
nitro group. Departure from coplanarity with the aromatic ring is also
pronounced with a -OR substituents, whereas an -OH group is nearly coplanar
[25].
1.6. Factor Affecting on Fluorescence Spectra:
1.6.1 Solvent Effect:
Although the fluorescence of gases [26-28] and solids [29] have been measured
the vast majority of fluorescence spectra have been measured in solution. As in
the case of ultraviolet absorption spectra [30], solvent effects are observed in
fluorescence measurements. These effects are manifested by wave length shifts
and/or quenching of fluorescence. Fluorescence solvent effects are less well
known than ultraviolet absorption solvent effects and the effects of pH on
absorption and fluorescence spectra; nevertheless, several groups of
compounds are known in which change of solvent brings about remarkable
changes. These changes are in some instances accompanied by comparable
changes in the fluorescence excitation spectra, and hence also in the ultraviolet
absorption spectra; in other instances shifts in the position of the fluorescence
13
emission maxima are not accompanied by comparable changes in the

ultraviolet absorption spectra. Displacements in the ultraviolet and fluorescence
emission spectra imply interaction with solvent in both the ground state and the
excited state of the molecule. On the other hand, when the fluorescence
emission spectra alone are affected by change of solvent, interaction between
solvent and the excited state, but not with the ground state of the molecule, is
indicated. An attempt will be made to discuss here the observed fluorescence
solvent effects in the light of accompanying changes, or lack thereof, in the
ultraviolet absorption spectra. As early as 1904 Kauffmann and Beisswenger
[31] observed changes in the fluorescence color of 3-aminophthalimide, I, with
change in solvent from violet in hydrocarbons to blue-green in ethanol.
Wavelength shifts brought about by solvent have been observed in both
absorption and fluorescence spectra of many compounds. The effect of the
dielectric constant of the solvent was used in explaining some fluorescence
solvent effects [32-35] and hydrogen bonding has been invoked in other
instances [36-38]. However, the interpretation of solvent effects is often
complicated because the observed changes may be the resultant of several quite
different forces which may either augment or minimize each other and these
effects have been discussed by various authors [39-43].Bayliss and McRae
[39,40] discussed the origin of solvent effects and point out that all spectra
undergo a polarization shift to longer wave length. This shift results from the
induced polarization of the solvent produced by the transition dipole of the
solute. It is more clearly observed in nonpolar solvents and solute, i.e., a red
shift occurs with increasing dielectric constant of the solvent. However, it is
often obscured by associated effects, e.g., in polar or hydrogen bonding
compounds. When the molecule changes to the excited state by the absorption
of energy this relationship is upset. An excited solute molecule in this situation
is in the Franck-Condon state. This state prevails for a very brief period of time
but the solvent molecules cannot reorient themselves during the transition from
the ground to the excited state. It follows then from the Franck-Condon
principle that there may be a difference in solvation energy of the ground and
14
excited states and this will be reflected in changes in absorption and emission
spectra. The “blue shift,” i.e., hypsochromic shift observed in absorption
spectra in solvents of high dielectric constant have been described [42,37] in
the light of the Franck-Condon principle. The blue shift is associated with
n → π * a transition which refers to the excitation of a nonbonding electron to
an antibonding orbital [43]. However, it was pointed out that n → π *
transitions are usually not observed in fluorescence spectra and when present
are weak [44].
1.6.2. Effect of pH:
The basicity or acidity of a molecule is determined by its electronic structure
and this may undergo detailed changes during excitation from the ground state
by the absorption of light, ie., the basicity or acidity of a compound may
change during excitation. A difference in the basicity or acidity of the ground
and excited states ill be reflected in differences between the absorption and
fluorescence spectra with change in pH. The nature of the changes will depend
on whether the basicity is increased or decreased during ionization. Also, in
order for the expected changes in pK to be observed, it is essential that the
ionization equilibrium be established during the lifetime of the excited state.
Weller [44] discussed the absorption and fluorescence spectral shifts in terms
of the energy changes involved. A simplified energy diagram is presented in
Fig.. This diagram represents the energy terms involved in the dissociation of
an acid (HA) and the corresponding base (B). ΔH represents the enthalpy of the
dissociation reaction in the ground state and ΔH* the same for the excited state.
When ΔH > ΔH* a red shiftoccurs in absorption and fluorescence spectra on
acid dissociation; when ΔH < ΔH* a blue shift occurs on acid dissociation, and
when ΔH = ΔH* there is no shift in either absorption or fluorescence spectra.
15
A*
HA* ΔΗ∗
ΔΕΑ
ΔΕΗΑ
A
ΔΗ
HA
Fig. 1.4. Energy diagram for dissociation of an acid (HA) in the ground
and excited state.
Since,
ΔH - ΔH* = ΔEHA - ΔEA (1)
where
ΔEHA = excitation energy for the 0-0 transition of the acid,
ΔEA = the excitation energy for the 0-0 transition of the base,
assuming that the entropy of the reaction of acid dissociation is the same for the
ground and the excited states it is possible to calculate the difference in pK
between the two states from equation 2.
Δ E HA − Δ E A
pK − pK *
=
2 . 303 RT
When
ΔE H A - ΔEA>0 the excited state is more acidic than the ground state, e.g., oxy-
and amino aromatic compounds; when ΔEH A - ΔEA < 0 the excited state is less
16
acidic, e.g., acridine and acridone, and, of course, when Δ E H A- Δ EA = 0 there

is no difference in pK between the ground and excited states, e.g., pyridine.
From the position of the 0-0 transition for acridine and the acridinium cation,
Weller, using the method described above, calculated the pK for the excited
state of acridine to be 10.35 and that for the ground state, 5.45. On this basis
then it is expected that in aqueous solution acridine will show a fluorescence
change at pH 10 which is independent of the absorption change at pH 5.45. The
green fluorescence of the cation should change to the blue fluorescence of the
free base between pH 9 and 11. Neutral aqueous acridine solutions, however,
fluoresce blue so that it has to be concluded that the required ionization
equilibrium is not established during the lifetime of the excited state.
1.6.3. Effect of Concentration:
Increases in concentration of fluorescent solutions usually result in quenching
at high concentrations and this is often accompanied by wave length 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 [45].
1.7. Fluorescence Quenching Phenomenon:
1.7.1 Fluorescence quenching:
The phenomenon of decrease in intensity of fluorescent compound at its
λmax 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. Further, the
quenching of one compound by another is often unpredictable [46-19]
17
Quenching of fluorescence can occur by-

a) Inner filter effect
b) Energy degradation
c) Chemical change
d) Energy transfer
e) 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
[10,37,38]
The two kinds of inner filter effects are excessive absorption of the
exciting light, and absorption of the luminescence emitted (solute quenching).
Therefore, the fluorescence intensity of mostly 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
only to an OD of about 0.05. Beyond these concentrations the fluorescence
intensities can be approximately corrected for the inner filter effects as fallows
Fcorr ≈ Fabs .anti log[(ODex + ODem ) / 2]
Many fluorescent dyes from dimmer or higher aggregates in solutions of

higher concentrations particularly in non-polar solvents. Often the energy
transfer [50, 51]
The second type of inner filter effect is that produced by the absorption of the
fluorescence light either by absorption of an excessive concentration of the
fluorescent dye itself (self absorption), or by other solutes. Self-absorption
mainly affects the low wavelength side of the fluorescence emission band
because it is in this region that overlap with the first absorption band occurs.
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.
18
1.7.2. 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 effects of oxygen
certain iodo, bromo and nitro compounds and probably may be due to energy
transfer. The quenching of fluorescent molecules by electron donating I-, Br-,
SCN-, and S 2 O3− and electron accepting anions such as IO3− , NO3− and S 4 O6− are
cases of involving electron transfer between donor-acceptor. The certain for
energy transfer should be that the triplet energy level; quencher should be lie
below the excited singlet level of fluorophor which is probable in condensed
system where donor-acceptor (quencher) are more closer than in solution
[52,53]
1.8.3. Quenching by energy transfer:
The system of donor – acceptor pair is excited by proper radiation which
selectively excited 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 by which donor deactivation
to its ground state and acceptor is raised to singlet excited level. The excited
acceptors so formed de-excites to ground level either radiatively or non-
radiatively. In non-radiatively process the fluorescence characteristics of
acceptor molecule is exhibited. The systematic variation of acceptor
concentration gradually quenches the fluorescence of donor by ET with
simultaneous sensitization of acceptor fluorescence. Quenching by energy
transfer can take place by two fundamentally different mechanisms [53-57]. In
the electron-exchange mechanism, two single independent electron –transfer-
one in each direction-result in formation of the sensitizer’s ground state and
quencher’s excited state. Energy transfer by dipole-dipole mechanism operates
by Coulombic resonance interaction (the “transmitter-antenna mechanism”), in
which the oscillating electrons of an excited-state sensitizer are coupled with
those of the quencher by an induced dipole interaction. Energy and electron
transfer processes, quenching mechanism shown in Scheme – 1. The photo-
19
physical and photo-chemical aspects of such systems are understood by the

kinetic studies of both fluorescence quenching and sensitization effect. The
analytical reactions are established in such studies to develop methods for
quantitative estimations of medicinal compounds such as vitamins,
erythromycin etc. [58-60]. Photo physical steps of energy and electron transfer
are shown in fig.
Fig.1.5. Photochemical steps of energy and electron transfer `

Energy and Electron transfer nomenclature
D+ + A D + A+ Hole transfer
D-+A D + A- Electron transfer
D*+A D + A* Energy transfer
1.8. Fluorescence quenching:

Quenching is any process that decreases the fluorescence intensity, and
as such, may be the result of a variety of processes. Some of these processes
are uninteresting, such as concentration changes or inner filter effects due to
excessive concentration or due to light scattering. Photobleaching, in which
high radiation intensity may damage the fluorophores, may also cause an
20
apparent quenching; the time-dependent nature of the process differentiates

photobleaching from normal quenching effects. One experimentally useful type
of quenching is due to collisions between quenching agents and fluorophores,
and is called collisional or dynamic quenching. A second type of quenching,
sometimes confused with dynamic 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 between 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:[61]
= 1 + k qτ 0 [Q] = 1 + K D [Q]
F0
F
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,
F0 τ 0
=
F τ
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:
f 0 = f Qk 0
21
where fQ is the fraction of collisions that result in quenching, and k0 is the

diffusion controlled bimolecular rate constant:
4πN 0
k0 = (r f + rq )( D f + Dq )
cm 3
1000
L
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 M-1sec-1. If the fluorophore is bound to the surface of a protein, the k0
will be roughly half of this value due 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:
[ FQ]
` Ks =
[ F ][Q]
where [FQ] is the complex concentration, and [F] and [Q] are the
concentrations of free quencher and free fluorophore. Because the total
fluorophore concentration, [F]0 = [F] + [FQ],
[ F 0] − [ F ]
Ks =
[ F ][Q ]
Which is rearrange
[ F ]0 1
Ks = −
[ F ][Q ] [Q ]
If you assume that the all of the decrease in observed fluorescence is due to
complex formation, the equation becomes:
F0
= 1 + K s [Q]
F
which is identical in form 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
22
lifetime, while static quenching merely reduces the apparent concentration of

the fluorophore. Alternatively, temperature effects can be used to distinguish
the two forms of quenching. Diffusion rates, and therefore dynamic quenching
rates, increase with higher 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 results in a modified equation: [62]
F0
= (1 + K D [Q])(1 + K s [Q]) = 1 + ( K d + K s )[Q] + K D K s [Q] 2
F
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: [25]
[ Q ]VN 0
F0
= (1 + K d [Q ])e 1000
F
in which V is the volume of the sphere (V is usually slightly larger than would
be predicted for 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 fluorophore.Assuming
the buried fluorophore is not quenched, and fluorescence will be:
23
F0 a
F= + F0 b
1 + K a [Q]
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 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 fluorophore to the quenching agent.
1.9. Fluorescence Resonance Energy Transfer (FRET) Process:
The use of Förster or fluorescence resonance energy transfer (FRET) as
a spectroscopic technique has been in practice for over 50 years. A search of
ISI Web of Science with just the acronym “FRET” returns more than 2300
citations from various areas such as structural elucidation of biological
molecules and their interactions, in vitro assays, in vivo monitoring in cellular
research, nucleic acid analysis, signal transduction, light harvesting and
metallic nanomaterials. The advent of new classes of fluorophores including
nanocrystals, nanoparticles, polymers, and genetically encoded proteins, in
conjunction with ever more sophisticated equipment, has been vital in this
development. This review gives a critical overview of the major classes of
fluorophore materials that may act as donor, acceptor, or both in a FRET
configuration. [63]
Fluorescence resonance energy transfer (FRET) is a nonradiative
process whereby an excited state donor D (usually a fluorophore) transfers
energy to a proximal ground state acceptor A through long-range dipole–dipole
interactions (Figure 1.7 and 1.8) [63-65]. The acceptor must absorb energy at
24
the emission wavelength(s) of the donor, but does not necessarily have to remit
the energy fluorescently itself (i.e. dark quenching).
Donor Fluorophore
S2
S1
Acceptor Fluorophore
ET
IC
Absorption
Emission
IC
S0 Emission
Fig.1.7 Mechanism of Energy Transfer in Donor and Acceptor
25
Fig.1.8. Schematic of the FRETprocess : Upon excitation, the excitedstate

donor molecule transfers energy nonradiatively to a proximal
acceptor molecule located at distance r from the donor. The
acceptor releases the energy either through fluorescence or
nonradiative channels. The spectra show the absorption (Abs) and
emission (Em) (63)
The rate of energy transfer is highly dependent on many factors, such as

the extent of spectral overlap, the relative orientation of the transition dipoles,
and, most importantly, the distance between the donor and acceptor molecules
[66]. FRET usually occurs over distances comparable to the dimensions of
most biological macromolecules, that is, about 10 to 100Å. Although
26
configurations in which multiple donors and acceptors interact are increasingly

common, the following equations consider energy transfer between a single
linked D/A pair separated by a fixed distance r and originate from the
theoretical treatment of Förster. [66-68] The energy transfer rate kT(r) between
a single D/A pair is dependent on the distance r between D and A and can be
expressed in terms of the Förster distance R0. R0 is the distance between D and
A at which 50% of the excited D molecules decay by energy transfer, while the
other half decay through other radiative or nonradiative channels. R0 can be
calculated from the spectral properties of the D and A species
9000 ln 10k 2 Φ D f (υ )ε (υ ) dυ
R06 =
128π 5 n 4 N ∫ υ4
Å
The factor k2 describes the D/A transition dipole orientation and can
range in value from 0 (perpendicular) to 4 (collinear/parallel). There has been
much debate about which dipole orientation value to assign for particular
FRET formats. Only in few cases can the crystal structure of the D/A
molecules be determined; there is no other reliable experimental method to
measure absolute or fixed k2 values, which leads to potential uncertainties in
subsequent calculations. [69-70] Fortunately, the accumulated evidence has
shown that the mobility and statistical dynamics of the dye linker lead to a k2
value of approximately 2/3 in almost all biological formats. This also sets an
upper error limit of 35% on any calculated distance.[66] Excellent discussions
of this issue are provided by dos Remedios and Moens[70] as well as
Stryer.[71] The refractive index n of the medium is ascribed a value of 1.4 for
biomolecules in aqueous solution. QD is the quantum yield (QY) of the donor
in the absence of the acceptor and J (λ) is the overlap integral, which represents
the degree of spectral overlap between the donor emission and the acceptor
absorption. The values for J (λ) and R0 increase with higher acceptor extinction
coefficients and greater overlap between the donor emission spectrum and the
acceptor absorption spectrum. Whether FRET will be effective at a particular
distance r can be estimated by the “rule of thumb” R0±50% R0 for the upper
and lower limits of the distance.[69,70] The efficiency of the energy transfer
27
can be determined from either steady-state [Eq. (2)] or timeresolved [Eq. (3)]
measurements.
FDA
E = 1− ------------- (2)
FD
τ DA
E = 1− ------------- (3)
τD
F is the relative donor fluorescence intensity in the absence (FD) and
presence (FDA) of the acceptor, and t is the fluorescent lifetime of the donor in
the absence (FD) and presence (FDA) of the acceptor. FRET is very appealing
for bioanalysis because of its intrinsic sensitivity to nanoscale changes in D/A
separation distance (proportional to r6). This property is exploited in FRET
techniques ranging from the assay of interactions of an antigen with an
antibody in vitro to the real-time imaging of protein folding in vivo.[72,73] The
myriad FRET configurations and techniques currently in use are covered in
many reviews.[69,74,75] Herein, we focus primarily on the fluorophore
materials utilized in bioanalytical FRET rather than the process itself. The
materials can be divided into various classes: organic materials, which includes
“traditional” dye fluorophores, dark quenchers, and polymers; inorganic
materials such as metal chelates, and metal and semiconductor nanocrystals;
fluorophores of biological origin such as fluorescent proteins and amino acids;
and biological compounds that exhibit bioluminescence upon enzymatic
catalysis. These materials may function as either FRET donors, FRET
acceptors, or both, depending upon experimental design.
1.10. Surfactant:
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 in dynamic association of surfactant molecules that achieve
segregation of their hydrophobic proteins from the solvent via self assembly [76].
28
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.1.9. The single 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. 1.9. Structure of Micelle (76)

The existence in one compound of two moieties, one of which has
affinity for the solvent and other of which is antipathetic to it, has been called
amphipathy by Hartlet [76]. 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.
29
1.11. CLASSIFICATION OF SURFACTANTS [77]:

From the commercial point of view surfactants are often classified according to
their use. However, this is not very useful because many surfactants have
several uses, and confusions may arise from that. The most acepted and
scientifically sound classification of surfactants is based on their dissociation in
water. The figures in page 4 show a few typical examples of each class.
Anionic Surfactant:
1.11.1. Anionic Surfactants:
Anionic Surfactants are dissociated in water in an amphiphilic anion,
and a cation, which is in general an alcaline metal (Na+, K+) or a quaternary
mmonium. 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) CH3(CH2)11SO-4 Na+

sulphate
3 Hexadecylsulphonic acid CH3(CH2)15SO-3 H+
4 Sodium
C8H17OOCHSO-3 Na+
dioctylsulphosuccinate
C8H17OOCCH2
1.11.2. Nonionic Surfactants:

Nonionic Surfactants come as a close second with about 45% of the
overall industrialproduction. 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
30
the polycondensation of ethylene oxide. 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 oposition to polyethylene oxide) is slightly
hydrophobic.
Sr. Name of the surfactant Structure
NO
1 Polyoxyethylene p-tertoctylphenyl C8H17C6H4O(CH2CH2O)10H
ether
2. Polyoxyethylene monohexadecyl CH3(CH2)15(OCH2CH2)21OH
ether
1.11. 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 ammoniums, with one or several long chain of the alkyl type, often
coming from natural fatty acids. These surfactants are in general more
expensive than anionics, 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 bactericide, (2) as
positively charged substance which is able to adsorb on negatively charged
substrates to produce antistatic and hydrophobant effect, often of great
commercial importance such as in corrosion inhibition. When a single
surfactant molecule exhibit 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 aminoacids and phospholipids.
31
Sr Name of Surfactant Structure

No
1 Hexadecyl (cetyl)trimethylammonium CH3(CH2)13N+(CH3)3 Br -
bromide
2
Dodecylpyridium chloride +
N
-
C12 H25 Cl
3 +
Dodecyl hydrochloride CH3(CH2)11NH3
1.11.4. Zweterionic or Ampholytic Surfactant:

This type can behave either as an ionic, non-ionic, or cationic species,
depending on the pH of the solution.
Name of surfactant Structure of surfactant
N-dodecyl-N:N-dimethyl betaine +
C12H25N (CH3)2CH2COO
-
1.12. 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” [76]. Surfactant molecules (e.g.
CTAB, SDS, Triton X-100, Brij – 35 etc.) self-aggregate into supremoleculear
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 [77-80]. A typical micelle has
size of ~50 Å and is made of about 100 surfactant molecules. [81] The self
association gives rise to rich variety of phase structure shown in fig.1.10.
Aggregation is not, however host limited to aqueous solution; it is some time
32
observed in non-aqueous polar solvents such as ethylene glycol and non-polar

solvents such as hexane. [82]
a. Spherical Micelle b. Elongated Micelle, c. Rod – shaped Micelle

d. Reverse Micelle
Fig.1.10. Different Phase Structure of Self – Association.

1.13. Solubilization of Organic Compounds:
Self association property of surfactants to form self – assembled
aggregates, the miclles, in an aqueous medium has a profound effect on the
solubility of the some Organic substance (Additives), otherwise sparingly
soluble in water [83-84]. Solubilization by micelles is of importance in many
33
industrial processes such as detergency, emulsion polymerization, oil recovery,

etc. and in a verity of fundamental research oriented polymerization like
micellar modeling of biological membrane. [85]
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 [86-89]. 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 [90] due to water dragging effect where the water is carried as a
shell around the solubilizate and water between the solubilizate and micelles,
and between water and micellar phase play a vital role in the partitioning
process. The partition coefficient is dependent on the structure of solubilizate
and the surfactant that constituent the micelles.
In addition to the solubilization equilibrium, the micro viscocity 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 visualization as the construction of
model membrane to mimic a biological surface with the solubilizate, drug
carrier and drug release [91, 92].
1.13.1. Solubilization:
Solubilization is believed to occur at a number of different sites in the
micelle as shown in Scheme-2 .
1. One is the surface of the micelle at the micelle solvents interface
2. Between the hydrophilic head group
3. In the so called 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
34
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 [93-94]. Surfactant micelles can be
pictured as having a highly nonpolar interior and a relatively polar interfacial
region. Therefore nonpolaized 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, replacing water
molecules that may have penetrate in to the core of the micelle close to the
polar heads, but solubilization of additional material is either deep in the
palisade layer as located in the inner core of micelle [84]. The polarzability of
the π electron cloud of the aromatic nucleus and its consequent ability to
interact wth the positively charged groups at the micelle water interface may
account for the initial adsorption of these hydrocarbons in that location
Scheme-2[95]
Scheme-2
35
1.14. Application:
The precise location and nature of the interactions between specific
molecular species in living cells is of major interest in many areas of biological
research, but investigations are often hampered by the limited resolution of the
instruments employed to examine these phenomena. Conventional widefield
fluorescence microscopy enables localization of fluorescently labeled
molecules within the optical spatial resolution limits defined by the Rayleigh
criterion, approximately 200 nanometers (0.2 micrometer). However, in order
to understand the physical interactions between protein partners involved in a
typical biomolecular process, the relative proximity of the molecules must be
determined more precisely than diffraction-limited traditional optical imaging
methods permit. The technique of Fluorescence Resonance Energy Transfer
(more commonly referred to by the acronym FRET), when applied to optical
microscopy, permits determination of the approach between two molecules.
Biosensors designed on the principle of fluorescent resonance energy
transfer (FRET) have been widely applied to visualize signaling cascades in
live cells with high spatiotemporal resolution. The application of FRET
biosensors in studying molecular events in live cells and analysis methods to
explore complex biological information implicated in FRET images
a) Determination of distance between donor and acceptor
The Forster resonance energy transfer can be used as a spectroscopic
ruler in the range of 10-100 Å. The distance between the donor and acceptor
molecules should be constant during the donor lifetime, and greater than about
10 Å in order to avoid the effect of short range interactions. Some precautions
must be taken to ensure correct use of the spectroscopic ruler as:
i) The critical distance R0 should be determined under the same
experimental conditions as those of the investigated system because
R0 involves the quantum yield of the donor and the overlap integral,
which both depend on the nature of the microenvironment.
ii) As regards the orientation factor k2, it is usually taken as 2/3, which
is the isotropic dynamic average, i.e. under the assumptions that both
36
donor and acceptor transient moments randomize rapidly during the

donor lifetime and sample all orientations.
b) Distributions of distances in donor-acceptor pairs
The donor-acceptor distance may not be unique, especially when the
donor and acceptor are linked by a flexible chain (e.g. end-labeled oligomers of
polymethylene oxides, polymer chains). The most commonly used method for
the evaluation of a distribution of distances is based on the measurement of the
donor fluorescence decay.
c) RET can be used to measure the extent of binding. The steady state
measurements are often used to measure binding interactions. It has efficient
application to study macromolecular systems when there is more than a single
acceptor molecule near a donor molecule.
d) Qualitative and quantitative applications of RET
In addition to the determination of distances at a supramolecular level,
RET can be used to demonstrate the mutual approach of a donor and an
acceptor at a supramolecular level as a result of aggregation, association,
conformational changes, etc. The donor and acceptor molecules generally
covalently linked to molecular, macromolecular or supramolecular species that
move toward each other or move away. Because of its simplicity, the steady
state RET-based method has been used in many diverse situations as shown
below.
i) In chemical sciences
• Polymers: interpenetration of polymer chains, phase separation,
compatibility between polymers, interdiffusion of latex particles,
interface thickness in blends of polymers, light-harvesting polymers
• Supramolecular systems: molecular devices, artificial photosynthesis,
antenna effect, etc.
• Restricted media and fractals. Morphology of porous solids.
Determination of dimensionality.
• Chemical sensors
• Scintillators
37
ii) In life sciences

• Ligand-receptor interactions
• Conformational changes of biomolecules
• Proteins: in vivo protein-protein interactions, protein folding kinetics,
protein subunit exchange, enzyme activity assays, etc.
• Membranes and models: membrane organization (e.g. membrane
domains, lipid distribution, peptide association, lipid order in vesicles,
membrane fusion assays, etc.)
• Drug-protein interactions
• Immunoassays
38
1.15. Literature Survey on Energy transfer and quenching Mechanism:

Yavuz Onganer et al. studied the fluorescence resonance energy
transfer from fluorescein to merocyanine 540 in aqueous sodium dodecyl
sulfate, cetyltrimethylammonium bromide and Triton X-100 micellar solutions
at room temperature using steady-state and time-resolved fluorescence
spectroscopy techniques.[96].
Markus Sauer et al studied the fluorescence resonance energy transfer
(FRET) efficiency of different donor–acceptor labeled model DNA systems in
aqueous solution from ensemble measurements and at the single molecule
level. The donor dyes: tetramethylrhodamine (TMR); rhodamine 6G (R6G);
and a carbocyanine dye (Cy3) were covalently attached to the 5′-end of a 40-
mer model oligonucleotide. The acceptor dyes, a carbocyanine dye (Cy5), and
a rhodamine derivative (JA133) were attached at modified thymidine bases in
the complementary DNA strand with donor–acceptor distances of 5, 15, 25 and
35 DNA-bases, respectively. Anisotropy measurements demonstrate that none
of the dyes can be observed as a free rotor; especially in the 5-bp constructs the
dyes exhibit relatively high anisotropy values. [97].
F. G. Sánchez et al reported the experimental study on the energy
transfer process between perylene and fluorescein in cetyltrimethylammonium
bromide (CTAB) micelles. The spectral behaviour and the dynamic and
polarization data of dyes confirmed that perylene is solubilized in the inner
hydrocarbon region of the micelle, while fluorescein micellized in the inner
part of the Stern region. The results obtained from the quenching experiments
demonstrated that a static mechanism operates for the perylene-fluorescein pair
in CTAB micelles. The study of the influence of the acceptor concentration on
the energy transfer efficiency suggested that only intramicellar energy transfer
contributes to the overall process. Moreover, the influence of the addition of
0.01 M NaOH on CTAB micelles was investigated by the changes in both the
critical micelle concentration (CMC) and the micellar aggregation number.
CMC values were obtained from the pyrene 1:3 ratio method, and micellar
39
aggregation numbers were determined by using a procedure based on the

energy transfer process. [98]
S. A. Stevenson and G.J. Blanchard have studied micelles comprised
of cationic (CTAB) and anionic (SDS) surfactants through the interactions of
solution phase anionic disodium fluorescein (DSF) and cationic rhodamine 110
(R110) dyes with perylene sequestered within the micelles. The efficiency of
this process is mediated by the extent to which the ionic dyes interact with the
micelle palisade layer, and their fluorescence lifetime data allow us to
determine the association constants for acceptor–micelle interactions [99].
J. Hadjianestis and J. Nikokavouras studied the spectral parameters
and quantum yield of luminal,3-aminophalate and fluorescein in cetyltrimethyl
ammonium chloride at various concentration, together with the solubilization
sites in the micelles. Chemiluminescence energy transfer to fluorescein results
in at least a tenfold increase in the intensity in the micellar system compared
with that in the absence of fluorescein. [100].
Kevin Toerne and Ray von Wandruszka shows that the Förster
energy transfer from excited surfactant species was used to monitor their
association with micellized perylene at different temperatures. Fluorescence
from the perylene acceptor was obtained through sensitization by surfactant
monomers and aggregates, the latter being identified as spectroscopic rather
than physical. Surfactant monomer fluorescence was subject to a strong inner
filter effect inside the micelles, indicating an abundance of spectroscopically
monomeric species there. Temperature increases caused reductions in native
surfactant fluorescence through thermal quenching, but sensitized fluorescence
intensities generally increased with temperature. This was ascribed to a
temperature-induced approach between the perylene acceptor and both
monomeric and aggregated surfactant donors. Clouding of the solutions
showed little evidence of organizational changes at the micellar level [101].
M Darbyshir et al report a new type of remote optical sensor of metal
ions in solution based on fluorescence lifetime quenching due to dipole-dipole
energy transfer across a polymer-water interface. Ion selectivity is provided by
40
spectral overlap between the sensor fluorescence and metal ion absorption. The
sensor is demonstrated in the detection of cobalt ions in water [102].
T. Salthammer et al reported the fluorescence quenching of perylene
by CoCI2"6HzO in small unilamellar DPPC vesicles via energy transfer. At the
probe-to-lipid ratio of 1:200 and quencher to lipid ratios of ≥ 12.5:1, donor-
donor energy transfer between clustered perylene molecules was observed as
well as energy transfer from the perylene molecules to cobalt ions both above
and below the main phase transition temperature of the lipid. The fluorescence
quenching of perylene by CoCl2.6H20 in the lipid gel state is shown to be well
described by förster long-range energy transfer when both donor-donor and
donor-acceptor energy transfer are considered. [103]
O. S. Wolfbeis et al described two new fluorescence resonance energy
transfer (FRET) compatible labels, their covalent linkage to oligonucleotides,
and their use as donor and acceptor, respectively, in FRET hybridization
studies. The dyes belong to the cyanine dyes, and water solubility is imparted
by a phosphonate which represents a new solubilizing group in DNA labels.
They were linked to amino-modified synthetic oligonucleotides via
oxysuccinimide (OSI) esters. [104].
A. Periasamy et al, have shown that the FRET is an ideal technique to
estimate the distance between interacting protein molecules in live specimens
using intensity-based microscopy. The spectral overlap of donor and
acceptor— essential for FRET—also generates a contamination of the FRET
signal [105]
S A. Stevenson et al, they are studied micelles comprised of cationic
(CTAB) and anionic (SDS) surfactants through the interactions of solution
phase anionic disodium fluorescein (DSF) and cationic rhodamine 110 (R110)
dyes with perylene sequestered within the micelles. Fluorescence lifetime
measurements monitor energy transfer between the nonpolar optical donor
within the micelle and ionic probes in the surrounding solution. The efficiency
of this process is mediated by the extent to which the ionic dyes interact with
41
the micelle palisade layer, and our fluorescence lifetime data allow us to
determine the association constants for acceptor–micelle interactions [106].
Interactions between the cationic polymethine dyes 3,3′-
diethyloxacarbocyanaine iodide (DiOC2), 3,3′-dioctadecyloxacarbocyanine
iodide (DiOC18), and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine
perchlorate (DiI) in the nano-scale volume of anionic sodium dodecylsulfate
(SDS) micelles have been studied using optical spectroscopy techniques. The
solubilization of pairs of dye molecules (DiOC18/DiI or DiOC2/DiI) within
SDS micelles was monitored by Forster resonance energy transfer (FRET)
between the dyes in each pair. The influence of the hydrophobicity of the dyes
DiOC2 and DiOC18 on the efficiency of their binding to SDS micelles and,
consequently, on the efficiency of FRET between DiOC18 and DiI or DiOC2
and DiI fluorophors has been analyzed. It has been shown that hydrophobic
interactions in addition to electrostatic interactions are of key importance for
the binding between the positively charged dyes and negatively charged
surfactant micelles [107].
Ququan Wang et al. shows the fluorescence resonance energy transfer
(FRET) model using two-photon excitable small organic molecule DMAHAS,
(trans-4-(N-2-hydroxyethyl-N-ethyl amino)-42-(dimethyl amino) stilbene) as
energy donor has been constructed and tried in an assay for avidin. In the
FRET model, biotin was conjugated to the FRET donor, and avidin was
labeled with a dark quencher DABS-Cl (4-(Dimethylamino) azobenzene-42-
sulfonyl chloride). Binding of DABS-Cl labeled avidin to biotinylated
DMAHAS resulted in the quenching of fluorescence emission of the donor,
based on which a competitive assay for free avidin was established.[108]
White-light emission is achieved from a single layer of diblock
copolymer micelles containing green- and red-light-emitting dyes in the
separate micellar cores and blue-light-emitting polymer around their periphery,
in which fluorescence resonance energy transfer between fluorophores is
inhibited due to micelle isolation, resulting in simultaneous emission of these
three species [109]
42
Fluorescence resonance energy transfer (FRET) in a cascade scheme

between three amphiphilic dyes 3,3′-dioctadecyloxacarbocyanine perchlorate
(DiOC18(3), donor), 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine
perchlorate (DiIC18(3), cceptor/donor) and 1,1′-dioctadecyl-3,3,3′,3′-
tetramethylindodicarbocyanine perchlorate (DiIC18(5), acceptor) has been
investigated at low dye concentration (10−5 mol/l) in water–micellar solutions
due to a forced assembling of dyes in nanoscale volume. The experimental data
have revealed that sodium dodecyl sulfate (SDS) micelles solubilize dye
molecules such that their hydrophilic heads are in contact with water, while
hydrophobic tails are embedded into the hydrocarbon core of the micelle.
FRET efficiency has been found to depend on the concentration of dyes in
micelles and the most effective when each SDS micelle. [110]
Kumar Pal et. al. studied structural and photophysical properties of
aggregated CdS quantum dots (QDs) capped with 2-mercaptoethanol in
aqueous medium. The hydrodynamic diameter of the nanostructures in aqueous
solution was found to be ~160 nm with the dynamic light scattering (DLS)
technique, which is in close agreement with atomic force microscopy (AFM)
studies (diameter ~150 nm). However, the UVvis absorption spectroscopy,
powder X-ray diffraction (XRD), and transmission electron microscopy (TEM)
studies confirm the average particle size (QD) in the nanoaggregate. The
steady-state and time-resolved photoluminescence studies on the QDs further
confirm preservation of electronic band structure of the QDs in the
nanoaggregate. To study the nature of the nanoaggregate we have used small
fluorescent probes, which are widely used as biomolecular ligands (2, 6-p-
toluidinonaphthalene sulfonate (TNS) and Oxazine 1), and found the pores of
the aggregate to be hydrophobic in nature. The significantly large spectral
overlap of the host quantum dots (donor) with that of the guest fluorescent
probe Oxazine 1 (acceptor) allows us to carry out Förster resonance energy
transfer (FRET) studies to estimate average donor-acceptor distance in the
nanostructure, found to be ~ 25 Å. carry out Förster resonance energy transfer
43
(FRET) studies to estimate average donor-acceptor distance in the

nanostructure, found to be ~25 Å..[111]
Jesper Wengel and co-workers has studied the detection of nucleic acid
hybridization via fluorescence resonance energy transfer (FRET) using pyren-
1-ylmethyl and perylen-3-ylmethyl N2′-functionalized 2′-amino-LNA
nucleosides incorporated into oligonucleotides exhibited a clear distance
dependence of the FRET efficiency, ranging from below 10% when the
fluorophores were approximately 40 Å apart to approximately 90% when the
fluorophores were in close proximity [112].
Sarah A. Stevenson and G.J. Blanchard, studied the micelles
comprised of cationic (CTAB) and anionic (SDS) surfactants through the
interactions of solution phase anionic disodium fluorescein (DSF) and cationic
rhodamine 110 (R110) dyes with perylene sequestered within the micelles.
Fluorescence lifetime measurements monitor energy transfer between the
nonpolar optical donor within the micelle and ionic probes in the surrounding
solution. The efficiency of this process is mediated by the extent to which the
ionic dyes interact with the micelle palisade layer, and our fluorescence
lifetime data allow us to determine the association constants for acceptor–
micelle interactions.[113]
J. C. Thomes, co-workers, measured the relative quantum yield of
fluorescence of proflavine bound to DNA as a function of the number of bound
dyes per nucleotide and the ionic strength allow the determination of the
binding constants and respective number of the two types of sites previously
postulated. It is demonstrated that 2-3% of the base pairs form sites where the
dye is strongly bound and fluoresces normally while in the other set of sites the
binding constant is 3-4 times weaker and the fluorescence completely
quenched. Comparison with complexes of Pro with double stranded [114].
1.16. Present work:
Fluorescence Resonance Energy transfers (FRET) is a nonradiative

process where by an excited state donor (D) transfer energy to a ground state
acceptor (A). FRET mainly occurs over distances comparable to most
44
biological macromolecules i.e. about 10 to 100 Å. The rate of energy transfer is

highly dependent on the extent of spectral overlap between the relative
orientation of the transition dipoles and distance between the donor and
acceptor molecules. It is a powerful technique for studying conformational
distribution and dynamics of biological molecules such as DNA, Protein etc,
which play key role in maintaining human health. Many of the biomolecules
and nutritional components of the diet need to be detected and analyzed.
During scrutiny of literature we found that the earlier methods used for
determination of biomolecule involved chromatographic separation followed
by spectrophotometry and fluorimetry analysis. Fluorescent Polynuclear
aromatic hydrocarbons such as perylene, anthracenes etc. are known as
efficient donor of energy in organic solvents. These methods involve laborious,
time consuming separations process prior to analysis. The work planned was to
study the photophysical properties of some biomolecules which are of
significant in human diet, Polynuclear aromatic hydrocarbons and dyes. The
spectral data obtained would found useful in deciding the donor (fluorescent
probe) and acceptor (biomolecules) pairs for FRET studies. One of the
objectives was to develop a technique based on FRET for determination of
biomolecules from nutrient or drugs without prior separation.
Many of the lotions and medicines such as vitamins. The effectiveness
of medicines depends upon their intermolecular interaction between drug and
biomolcules. The ground and excited state molecular interaction can be
understood by absorption and fluorescence quenching spectroscopy involving
energy transfer or charge transfer between drug and biomolecules. The
attempts are made to study the interaction using suitable donor-acceptor pair of
drug and biomolecule.
The quenching experiments were set systematically with a view to
obtain the gradual quenching of donor fluorescence with successive additions
of acceptor solution. The validity of stern-volmer equation for the observed
biomolecular fluorescence quenching was tested and the quenching rate
constants were determined to known efficiency of energy transfer process. The
45
selective excitation of donor and fluorescence quenching data helped to

establish analytical relation between extent of quenching and acceptor
concentration which is used further for determination of acceptor in medicinal
tablets. The quenching studies were performed in micellar solution to provide
close approaches between donor-acceptor pair and to enhance energy transfer
process. The pharmokinetic studiesperformed in present work was given to
information regarding critical energy transfer distance and distance of
separation between centers of donor and acceptor molecules in the aqueous and
micellar solution.
46
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CHAPTER 1-Luminescene Topics

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CHAPTER – I Introduction

that the emission spectrum is typically a mirror image of the absorption

108 s-1, so that a typically fluorescence lifetime is near 10ns. Fluorescence

1.3. Processes competing with Fluorescence:

Fig.1.2. De-Excitation pathway of excited Molecules

1.4. Experimental Observables:

Fig. 1.3. Illustration of the vibrational bands in the absorption and

Extensive work has been carried out to establish exact relationship

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

The excitation spectrum will be identical to the absorption spectrum

1.4.4 Fluorescence Quantum Yields:

The quantum efficiency of fluorescence Φf is defined as the ratio of the

The most direct method for determination of fluorescence efficiency

The integrated area under the corrected fluorescence spectrum is

1.5.1. Electron-withdrawing substituents: (carbonyl and nitro compounds)

The fluorescence properties of aromatic carbonyl compounds are

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

Some aromatic carbonyl compounds have a low-lying π → π * excited

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

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

emission maxima are not accompanied by comparable changes in the

acidic, e.g., acridine and acridone, and, of course, when Δ E H A- Δ EA = 0 there

Quenching of fluorescence can occur by-

Many fluorescent dyes from dimmer or higher aggregates in solutions of

1.7.2. Quenching by energy degradation:

physical and photo-chemical aspects of such systems are understood by the

Fig.1.5. Photochemical steps of energy and electron transfer `

1.8. Fluorescence quenching:

apparent quenching; the time-dependent nature of the process differentiates

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

lifetime, while static quenching merely reduces the apparent concentration of

Fig. 1.6. Distinction between dynamic and static quenching.

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

Fig.1.7 Mechanism of Energy Transfer in Donor and Acceptor

Fig.1.8. Schematic of the FRETprocess : Upon excitation, the excitedstate

The rate of energy transfer is highly dependent on many factors, such as

configurations in which multiple donors and acceptors interact are increasingly

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

Fig. 1.9. Structure of Micelle (76)

1.11. CLASSIFICATION OF SURFACTANTS [77]:

Sr. No Name Structure

2 Sodium dodecyl (lauryl) CH3(CH2)11SO-4 Na+

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

1.11.2. Nonionic Surfactants:

the polycondensation of ethylene oxide. They are called polyethoxylated

1.11. 3. Cationic Surfactants

Sr Name of Surfactant Structure

1.11.4. Zweterionic or Ampholytic Surfactant:

1.12. The micellization process:

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

a. Spherical Micelle b. Elongated Micelle, c. Rod – shaped Micelle

Fig.1.10. Different Phase Structure of Self – Association.

industrial processes such as detergency, emulsion polymerization, oil recovery,

Ionic micelles ordinarily have an extensive hydrophobic core region,

donor and acceptor transient moments randomize rapidly during the

ii) In life sciences

1.15. Literature Survey on Energy transfer and quenching Mechanism:

aggregation numbers were determined by using a procedure based on the

Fluorescence resonance energy transfer (FRET) in a cascade scheme