Chapter 1

Introduction

Chapter 1

1.1 Luminescence :

1.1.1 Brief history of luminescence:

Our heritage from ancient times has been knowledge of luminescence in

the form of fireflies, glowworms, the lantern fish (Lucerna piscis), Nile fish

(dilyxnos), bacteria, rotten wood, fungus, mushrooms, mollusc, squid, etc. In

addition, the ancients had noted luminescence connected with electrical

phenomena such as the aurora borealis which are northern and southern polar

lights displays in sky usually observed at night, ignis lambens and pollux. The

fairly wide knowledge of luminescence begin with Aristotle (384-322 B.C.) in

Greece [1]. He observed luminescence of flesh, fish and wooden materials. The

Greek geographer Strabo (63 B.C. to 24 A.D.) mentioned dilyxons as a Nile

fish. Nicolas Monardes (1565) observed the light emission from an infusion of

plant Lignum Nephriticum. The emission of light in solids was discovered by

Bolognian Vincenzo Cascariolo (1603) in the form of Bolognese stone which

glows after exposure to light. Robert Boyle (1667) was the first to make

chemical experiments on bioluminescence. He worked on shining wood, fish,

certain, bacteria, fungi as well as on thermoluminescence [2]. Benjamin

Franklin (1752) identified the luminescence of lightning. The red emission of

chlorophyll was noted by Brewster in 1883. Besides of these, Arthanasius

Kricher (Germany), Francesco Grimaldi (Italy), Issac Newton (England), all

have observed the phenomenon of luminescence but they could not explain the

reasons behind it. The first detailed paper on luminescence was in 1852 by Sir.

G. G. Stokes (England) described the theoretical basis for the technique by

giving a mechanism of the absorption and emission process and termed the

phenomenon as fluorescence because the specimens used were of the fluorspar

mineral [3,4].

The term ‘Luminescence’ was first used in 1888 by German physicist

and historian of science Eilhardt Wiedemann. He defined luminescence as ‘all

those phenomena of light which are not solely conditioned by the rise in

temperature’. Wiedemann recognized luminescence as the contrast of

1

Chapter 1

incandescence where luminescence refers to cold light and incandescence

refers to hot light.

Wiedemann basically classified luminescence into six classes-depending

upon method of excitation. By using modern terminology this classification can

be extended into following classes as given in Table 1.1

Table 1.1: Types of luminescence:

Sr. No.

Type of Luminescence

Mode of Excitation

1 Photoluminescence (Fluorescence, Phosphorescence)

UV-Visible light

2 Thermoluminescence Heating after prior storage of energy.

3 Electroluminescence Electrical field

4 Crystalloluminescence Crystallization from solutions

5

Triboluminescence (Piezoluminescence )

Frictional and electrostatic forces

6

Chemiluminescence Chemical reactions

7

Galvanoluminescence Passage of electric current through aqueous solutions

8

Sonoluminescence Intense sound waves

9

Lyoluminescence Dissolution of crystals

10

Bioluminescence Biochemical reactions

11

Radioluminescence Particles emitted from radioactive material

12

Roentgenoluminescence High energy x-rays

13

Cathodoluminescence Cathode rays

14

Ionoluminescence Positive or negative ions

15

Anodoluminescence Anode rays

2

Chapter 1

Luminescent compounds are of very different kinds. Some of them can be

listed as follows:

a) Organic compounds: Aromatic hydrocarbons, coumarins, polyenes.

oxazines, fluorescein, amino acids, etc. b) Inorganic compounds: Uranyl ion (UO2

+), lanthanide ions, glasses with Nd,

Mn, Ce, Sn, Cu, Ag, etc, crystals like ZnSe ZnS,

CdS, CdSe, GaS, GaP, Al2O3, etc.

c) Organometallic compounds: Lanthanide ion complexes, Ruthenium

complexes, complexes with fluorogenic

chelating agents like 8-hydroxyquinoline, etc.

1.1.2 Origin of Photoluminescence:

Photoluminescence is one of the major classes of luminescence in which

substance absorbs photons and attains excited state. Consequently it re-emits

photons to return ground state. The emission of photons accompanying de-

excitation is called photoluminescence. It comprises mainly fluorescence and

phosphorescence.

The first reported observation of fluorescence was made by Spanish

physician Nicolas Monardes in 1565. He described wonderful blue color of an

extract of a wood called Lignum Nepriticum. This phenomenon was further

studied by G. G. Stokes by performing experiments with the solution of quinine

sulphate. Initially he called this phenomenon as ‘dispersive reflexion’ but after

words renamed as fluorescence [5, 6].The name ‘fluorescence’ originates from

‘fluorspar’ or ‘flurospath’ which are minerals containing calcium fluoride and

exhibit the phenomenon of fluorescence.

The term phosphorescence comes from Greek word ‘phosphor’ meaning

‘which bears light’. The term phosphor has been assigned from ancient periods

for those materials which glow in the dark after exposure to light. This property

was earlier reported by V. Cascariolo (1602) from Bologna for the bolognian

phosphor. In early times fluorescence and phosphorescence seems to be

identical as both are relevant to photoluminescence. The distinction between

fluorescence and phosphorescence based on experimental facts was made in

3

Chapter 1

nineteenth century. Fluorescence is an emission of the light which disappears

with end of excitation and in phosphorescence the emission persists after the

end of excitation. The first theoretical distinction between these two

phenomena was provided by Francis Perrin [7].

The twentieth century and specially the period of 1918-48 was prolific

for development of the major experimental and theoretical concepts of

fluorescence and phosphorescence. J.Perrin, Stern, Volmer, F.Weigen, S. J.

Vavilov, W. L. Levshin, F. Perrin, E. Gaviola, E. Jette, W. West, A. Jablonski,

Th. Förster are some of the names who made their efforts to make the concept

of photoluminescence more and more clear [8].

Nowadays there has been tremendous growth in the use of fluorescence

in various branches of science. Fluorescence spectroscopy and time resolved

fluorescence are considered important research tools in biochemistry and

biophysics. Fluorescence is leading technology used extensively in medical

diagnostics, biotechnology, drug analysis, flow cytometry, DNA sequencing,

forensics, genetic analysis, etc. The biochemical application of fluorescence

generally includes anisotropy measurements, resonance energy transfer, etc.

Anisotropy measurements provide information on the size and shape of the

proteins. It has been used to measure protein-protein associations and fluidity

of membranes. Resonance energy transfer has been used to investigate the

binding interactions and to measure molecular distances. The measurements

can provide information on a wide range of molecular process. Fluorescence

spectroscopy will continue to contribute to advancements in biology,

biotechnology and nanotechnology.

1.2 Light and its interaction with matter:

1.2.1 Nature of light:

An electromagnetic radiation, which generally spoken as light, is

characterized by a wavelength (λ) and frequency (υ) which are interrelated as ,

λυ c= (1.1)

where c is the velocity of light = 3 x 1010 cm/s

4

Chapter 1

After striking with matter, light can either pass through matter without

any absorption or it can be absorbed by matter entirely or partially. The energy

from light is absorbed in integral units, called quanta or photon. The

relationship between quanta and energy is given by,

λυ hchE == (1.2)

where E is the energy associated with photon and h is Planck’s constant.

Each molecule has a series of closely spaced energy levels and

absorption of a discrete quantum of light results the jump from lower to higher

energy level.

The mechanism of photoluminescence is based upon absorption and

emission of photon from electromagnetic radiation. It is divided mainly into

two categories fluorescence and phosphorescence depending on the nature of

the excited state. There are two possible arrangements in excited state as shown

in following Figure 1.1

Figure 1.1: Distinction between singlet and triplet excited states.

In ground state, two electrons are paired with each other and according

to equation, M = 2S + 1, where M is multiplicity and S is total spin quantum

number, the multiplicity of ground state is singlet. After absorption of photon,

two probable excitation states can be observed.

5

Chapter 1

(a) the electron in the excited orbital is paired to second electron in the ground

state orbital resulting into singlet multiplicity and hence called as excited

singlet states which are denoted as S0, S1, S2 , etc.

(b) the electron in the excited orbital is parallel to second electron in the

ground state orbital resulting into triplet multiplicity and hence called as

excited triplet states which are denoted as T1, T2, T3, etc.

The absorption of radiation is highly specific and radiation of particular

energy is absorbed only by a characteristic structure.

1.2.2 Fluorescence and Phosphorescence:

On the basis of excited state involved in absorption and emission

process, fluorescence and phosphorescence can be distinguished as follows:

a) Fluorescence

When molecule is in excited state, some energy, in excess, of the lowest

vibrational energy level is rapidly dissipated. If all the excess energy is not

further dissipated by collisions with other molecules the electron returns to the

ground state, with emission of energy. This phenomenon is called as

fluorescence. It involves the transition from lowest excited singlet to ground

singlet state. Because some energy is lost in the short period before the

emission, the fluorescence is of longer wavelength than the energy that was

absorbed. Generally the time period of fluorescence is 10-8 sec.

b) Phosphorescence

The phosphorescence involves the transition from excited singlet state to

excited triplet state and then from excited triplet state to ground singlet state.

This process is highly improbable as it is forbidden process because it involves

electron spin reversal. The characteristic transition times of phosphorescence

are 10-4 to 10 sec. It involves afterglow i.e. emission continues even after the

excitation source is removed. This is because of the relatively long life time of

the triplet state.

1.2.3 Mechanism of fluorescence with Jablonski diagram:

The mechanism of fluorescence can be well explained with the classical

Jablonski diagram, proposed by Professor Alexander Jablonski in 1935 to

6

Chapter 1

describe absorption and emission of light. Prof. A. Jablonski is known as

‘Father of Fluorescence Spectroscopy’ because of his versatile contribution to

the branch of fluorescence spectroscopy studies including descriptions of

concentration depolarization and defining the term ‘anisotropy’ to describe the

polarized emission from solutions [9,10]. The Jablonski diagram has many

forms, one such classical diagram is as shown in Figure 1.2.

The various singlet ground and excited are denoted by S0, S1, S2,........... etc. At each of these electronic energy levels the molecule can exist in a

number of vibrational energy sublevels. Similarly various triplet excited states

are denoted by T1, T2, T3,…. etc.

Following light absorption, which take place usually in about 10-15 sec,

several processes can occur. A molecule is excited to some higher vibrational

level either S1 or S2. The molecule in higher excited state rapidly relax to the

lowest vibrational level of S1. This process is called as internal conversion (IC)

and generally occurs in 10-12 sec. Consequently transition from S1 to S0 is called

fluorescence. Since fluorescence life times are near 10-8 sec, internal

conversion is generally complete prior to emission. Hence fluorescence

emission generally results from the thermally equilibrated excited state. As for

absorption, the electronic transition down to the lowest electronic level also

results in an excited vibrational state. This state will also reach thermal

equilibrium in about 10-12 sec.

The molecule in S1 state can undergo conversion to the first triplet state

T1 which is termed as intersystem crossing (ISC). The emission, following by

transition from T1 to S0 , is called as phosphorescence. It is generally shifted to

longer wavelengths relative to fluorescence. The transition from T1 to ground

state is forbidden and as a result the rate constant for such emission is smaller

than those of fluorescence.

1.2.4 Types of fluorescence:

The fluorescence emission from S1 state is referred to as prompt or

steady state fluorescence and it persists until the excitation is in process. As

soon as the excitation is stopped, the fluorescence emission cuts off. The

7

Chapter 1

phosphorescence is long lived delayed emission having spectral characteristic

very different from fluorescence. However, there are delayed emission whose

Figure 1.2: Jablonski diagram

spectra coincides exactly with prompt fluorescence from lowest singlet state

with only difference being in their lifetimes. These processes having lifetime

property of phosphorescence and spectral properties of prompt fluorescence are

known as delayed fluorescence [11].

1.2.4.1 E-type delayed fluorescence:

When excitation source is cut off, the excited triplet molecules do not

emits immediately due to its longer lifetime. If the energy gap between first

excited singlet and triplet ( ) is comparably smaller as in the case of dye

molecules, then the back energy transfer from triplet to singlet can occur. In

this sequence, the triplet excited molecule in the lowest vibrational level

acquire some thermal energy to go into the vibrational level of isoenergetic

with lowest vibrational level of first singlet state (S

11 TSE −Δ

1). This is followed by

energy transfer between isothermal vibrational level of triplet and first excited

singlet state. The first excited singlet state, consequently deactivates with the

8

Chapter 1

emission of light. This type of fluorescence was first observed in deoxygenated

solutions of eosin in glycerol and ethanol and hence it is referred as E-type

delayed fluorescence. Subsequently similar type of fluorescence was observed

from dyestuffs in fluid solutions [12].

1.2.4.2 P-type delayed fluorescence:

If energy gap between singlet and triplet state (ΔES ـT) is large, the

population of excited singlet state through back energy transfer is not possible.

In such cases, lowest excited singlets are formed in triplet-triplet annihilation

process. The emission occurred from lowest excited singlet state during

deactivation is termed as P-type delayed fluorescence as it was first observed in

pyrene and phenanthrene solutions.

1.3 Photophysical processes:

A photophysical process is defined as a physical process resulting from

the electronic excitation of a molecule or system of molecules by non ionizing

electromagnetic radiation. There are number of photophysical processes to

occur with interaction between radiation and molecule which are mainly

classified as:

1) Unimolecular processes and 2) Bimolecular processes.

1.3.1 Unimolecular processes:

The unimolecular photophysical processes that can occur in an isolated

molecule in the vapour phase at low pressure or in dilute solution in a

transparent medium can be divided into following categories:

a) Radiative excitation (absorption) transitions-

In this process, the molecule is excited from a lower to higher electronic

state by the absorption of photon.

These transitions are sub divided as

i) S0-S1and S0-Sp (P>1) i.e. the absorption from ground singlet state to

any excited singlet state is spin allowed and corresponds to main

electronic absorption spectrum.

9

Chapter 1

ii) S0-T1 and S0-Tq (q>1) i.e. the absorption from ground singlet state to

any excited triplet state is spin forbidden and can be observed with

intense light sources or perturbation methods.

iii) T1-Tq (q>1) i.e. the absorption from triplet state to higher triplet state

which commonly observed by flash photolysis. T1 is populated by

intersystem crossing from S1 which initially excited by an intense

flash light.

iv) S1-Sp (p>1) i.e. the absorption from first excited singlet state to

higher excited singlet state which observed by nanosecond flash

photolysis [13].

b) Radiative de-excitation (luminescence) transitions-

In this process, the molecule is deactivated from a higher to a lower

electronic state by emission of a photon. A radiative transition between states

of the same multiplicity is described as fluorescence while that between states

of different multiplicity is termed as phosphorescence.

These transitions are subdivided as

i) S1-S0 The fluorescence of short duration (~1-103 ns) corresponds to

the normal fluorescence emission.

ii) T1-S0 The phosphorescence which generally occurs with long

duration as it is spin forbidden.

iii) Sp-S0 Such fluorescence is observed in few compounds like azulene

[14].

iv) Tq-S0 This phosphorescence is very improbable process reported in

fluoranthene .

v) Tq-T1 The fluorescence corresponding to the inverse of T1-Tq

absorption reported in azulene and naphthalene [15].

c) Radiationless transitions

These involves transitions between isoenergetic vibrational levels of

different electronic state. Such transitions are normally proceeded by

radiationless thermal activation of the initial electronic state and/or followed by

raditaionless thermal deactivation of the final electronic state. A radiationless

10

Chapter 1

transition between states of the same multiplicity is termed as internal

conversion while that between state of different multiplicity is termed as

intersystem crossing. These transitions are subdivided as[16]

i) S2-S1 and Sp-Sp-1 The internal conversion process usually occurs

rapidly.

ii) T2-T1 and Tq-Tq-1 The internal conversion process usually occurs

rapidly.

iii) S1-S0 The internal conversion to the ground state.

iv) S1-T1 and S1-Tq The intersystem crossing constitutes the internal

quenching of S1 which competes with the normal fluorescence.

v) T1-S0 The intersystem crossing competes with the normal

phosphorescence.

vi) T1-S1 The intersystem crossing may occur by thermal activation of

T1 during its excitation lifetime to a vibrational level isoenergetic

with S1. This leads to E-type delayed fluorescence.

vii) Sp-Tq The intersystem crossing from higher excited singlet states.

1.3.2 Bimolecular processes:

The processes which occur in concentrated or aggregated systems due to

interactions with molecules of the same species (homopolar) or in mixed

molecular systems due to interactions with molecules of different species

(heteropolar) are called bimolecular processes.

The various bimolecular processes are divided into following categories:

a) Perturbation processes:

The interaction with an adjacent molecule may perturb the energy levels

of the excited molecule and modify its photophysical properties and behaviour.

b) Excitation migration:

The interaction between excited and unexcited molecules can lead to the

transfer of its excitation energy either by a radiative process or by a

radiationless process.

11

Chapter 1

c) Complex formation by aromatic molecules:

The heteropolar donar–acceptor complexes are formed between

aromatic hydrocarbons and other appropriate molecules in the ground state and

photophysical properties of these complexes which may be fluorescent or

phosphorescent ,differ from those of their constituent molecules.

d) Complex formation by an excited molecule and an unexcited molecule:

Many molecules in their first excited singlet state ( 1M*) interact with

unexcited molecules(1M) of same species to produce excited dimer (1D*)

1M* + 1M → 1D*

These homopolar excited dimeric complexes are known as excimers.

The excimers are distinct molecular species which exhibits its own

characteristic photophysical properties .

Similarly an excited molecular complex of definite stoichiometry which

is dissociated in the ground state is described as an exciplex. The exciplex

(1E*) is formed by the interaction of molecules in excited singlet state of one

species (1M*) with unexcited molecules of another species( 1Q) 1M* + 1Q → 1E*

e) Interaction between two excited molecules:

This type of interaction is to be observed between two identical

molecules each in the excited triplet state ( 3M*) 3M* + 3M* → 1D*

or 3M* + 3M* → 1M* + 1M

This process yields both excited molecules( 1M*) and excimer ( 1D*) and the

fluorescence of these entities involves P-type delayed fluorescence.

1.4 Processes competing with fluorescence:

The number of photophysical processes occurs as a result of interaction

between matter and radiation as discussed earlier. Though these processes

seems to be quite easily occurring, there are non-radiative processes which

precede or compete with fluorescence. Hence in accordance with the study of

fluorescence, it is important to consider the processes which competes with

fluorescence.

12

Chapter 1

1.4.1 Vibrational relaxation:

It is assumed that at room temperature, before excitation, all molecules

are in the lowest vibrational levels of the ground electronic state. By absorbing

radiation, a molecule is excited to one of the vibrational level of excited

electronic state. After arriving in the excited state, the excited molecule may be

in vibrationally excited state. Then the molecule will start to vibrate with a

characteristic frequency of that state, loosing its excess vibrational energy in

the form of infrared quanta or in the form of kinetic energy imparting to other

colliding molecules. Thus the excited molecule gets relaxed thermally to the

lowest vibrational level of the electronically excited singlet state. In gaseous

state, the deactivation of molecule from same vibrational level to which it is

excited occurs but in the solids and solutions, the excited molecule have to fall

into lowest vibrational level of the excited state before to deactivate. This

process of dissipation of energy in the form of heat and vibrations is known as

vibrational relaxation having life time of 10-14 to 10-12 sec. In this non-radiative

process, the molecule fall into the lowest vibrational level of an excited state

and then emission occurs as stated by Kasha’s rule. Hence when fluorescence

from solution occurs, it involves a transition from the lowest vibrational level

of an excited state [17]. Due to vibrational relaxation, the fluorescence band for

given electronic transition is shifted towards longer wavelengths form the

absorption bands.

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.

Internal conversion can be achieved in one of three possible ways:

a) If there is considerable overlap between the lower vibrational level of

the higher electronic state and higher vibrational level of the lower

electronic state then the upper and lower electronic states will be in

13

Chapter 1

transient thermal equilibrium. Then the molecule crossover from higher

to a lower excited singlet state by this vibrational coupling.

b) If there is no considerable overlap but they separated by a small gap,

the molecule in the upper electronic state will convert to the lower

electronic state by tunneling mechanism.

c) If the energy separation of the upper and lower electronic states are

relatively large, the radiative transition takes place to any one of a

number of vibrational levels of the lower electronic state. This radiative

transition is nothing but the fluorescence .

Internal conversion is very rapid process taking about 10-12 sec. The

average lifetime of the lowest excited singlet state is of the order of 10-8 sec .

Therefore even if a molecule can not pass efficiently from its lowest excited

singlet state to the ground state, it may undergo other processes which may

compete with fluorescence [18].

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. This is spin dependent internal conversion which may be fast

enough taking about 10-8 sec.

For efficient transfer to triplet state , molecule should have to satisfy

following conditions:

a) the energy difference between the lowest singlet state and the triplet

state, just below it, must be small.

b) vibrational coupling should be more between the excited singlet state

and triplet state.

In aromatic hydrocarbons where the singlet-triplet splitting is large, the

ISC is less efficient than in certain dye molecules where triplet-triplet

splitting is small [19]. As ISC occurs, subsequently the molecule undergoes

the IC process and falls to the lowest vibrational level of the first excited

triplet state. Therefore ISC can compete with fluorescence and this it

decreases the quantum efficiency of fluorescence. The population of triplet

14

Chapter 1

state has a significance in producing delayed fluorescence and

phosphorescence, which is radiative decay of triplet state molecule to the

ground state.

The possible de-excitation pathways are summarized in following Figure 1.3

Figure 1.3: Possible de-excitati

on pathways of excited molecules.

1.5 Ch racteristics of fluorescence:

he phenomenon of fluorescence has a number of general characteristics.

The fluorescence spectrum recorded is a fluorescence intensity as a function of

wavelength. The wavelength at which maximum emission takes place is

referred as emission wavelength (λem) while the height of emission peak at λem

gives intensity of fluorescence (F).

1.5.1 Fluorescence intensity:

he intensity of fluorescent light (F) is directly related with the

concentration of fluorescent solute in solution as given by the relation,

F =( I – I ) Φ (1.3)

is

fficient ε of the molecule [20,21].

1.5.2 F

a

T

T

0 F

If ΦF is constant then the shape of the fluorescence spectrum

determined solely by the extinction coe

actors influencing the fluorescence intensity:

Fluorescence intensity of a compound is altered by following factors-

a) Structure of the compound:

15

Chapter 1

It is observed that all organic compounds are not fluorescent but those

which exhibit fluorescence are usually aromatic or contain conjugated double

electronic phenomenon, the molecules having

readily

ne-pair’

electrons.

these

d compound is likely to

be mo if

e the π-electrons, there will be a diminution of

fluores

re fluorescent than benzene and its derivatives. Naphthalene,

anthrac

of the compound to be assayed is very important consideration in

quantit

at high dilutions where the

l enough to make the extent of re-

bonds. As fluorescence is an

available electrons for energy transitions are capable to show

fluorescence. Such electrons are π or delocalized electrons and ‘lo

If a compound contains π-electrons, there is good possibility that it will

show fluorescence and if a substituent, increasing the freedom of

electrons, is added to the compound, then the substitute

re fluorescent than the parent compound [21,22]. On the other hand ,

the substituent tends to localiz

cence.

For example, cyclohexane having no conjugated double bands is non –

fluorescent while benzene, an aromatic compound is weakly fluorescent. In

polycyclic aromatic systems, the number of π-electrons available is greater than

in benzene and therefore these compounds and their derivatives are usually

much mo

ene and biphenyl derivatives [23,24] are much more fluorescent than

the corresponding fluorescent benzene derivatives.

b) Concentration of fluorescent solute:

The intensity of fluorescence is proportional to the concentration of the

fluorescent compound only in highly dilute solutions and therefore the

concentration

ative work [25]. In most fluorimeters, the fluorescence emitted from the

cell holding the solution is measured at right angles to the path of exciting light.

The fluorescence emitted has therefore to pass through the solution to the

detector and during this passage some of it is re-absorbed by other molecules of

the compound under study. Higher the concentration of the compound, greater

is the proportion of the re-absorption. Therefore, linearity between fluorescence

intensity and concentration can only be expected

number of molecules present is smal

16

Chapter 1

absorption trifling compared with the amount of fluorescence emitted.

However, the effect of concentration is dependent to some extent upon

instrumental parameters such as slit width, intensity of the exciting light and

the type of detector. To get reproducible results, slit width and light intensity

need to keep constant during fluorimetric assay.

c) Effect of solvent:

The solvent used for fluorimetric analysis can affect the intensity and

wavelength of fluorescence. The solvent effect can be discussed into three

aspects-

i) Purity of solvent:

Since fluorimetry is a highly sensitive technique, it is important

that the solvent used should themselves be non-fluorescent and free

from fluorescent impurities. These solvents may be used either for

extracting the desired materials or for the actual fluorescence

measurements. Apart from water, number of solvents including

methanol, butanol, ether, hexane, heptane etc may be used for

elated work.

sorbs ultraviolet light and hence it is preferable to clean cuvettes in

chromic acid.

ii) Non

:

of the buffer affect the fluorescence. For example, in case of phosphate

fluorescence r

All solvents should be free from contaminants which may enter

through cleansing agents of glassware. For example, chromic acid

ab

nitric acid rather than

-aqueous solvent:

The fluorescence wavelength can alter depending upon the

physical properties of solvent such as dielectric constant, the association

of solvent and solute by hydrogen bonding, etc. For example, the reports

on indole shown that, the fluorescence wavelength increases with the

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

iii)Aqueous buffer solutions

The fluorescence measurements are carried out in aqueous buffer

solutions. In such cases, it is important to know whether the constituent

17

Chapter 1

buffer, an increase of phosphate concentration frequently leads to

diminution in fluorescence intensity.

d) pH

The ef

import

may b

fluores

range of

the pH-fluorescence change. In such

indicators, flu

e) Tem

decrea

molecu ndency for collisions. This would

some of the energy which might have radiated as

same time cause

decomposition. However, even though photo-decomposition

e two characteristic spectra: the excitation

ission spectrum. The large molecule have large number of

electrons and nuclei hence the absorbed energy can be readily distributed

of the solution:

fect of pH upon the fluorescence of a compound is of more

ance. A compound may be fluorescent over limited range of pH and it

e practically non–fluorescent in remaining pH regions. Again it may be

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

it may be much more fluorescent than over the rest, the working

fluorescent indicator is based on

orescence is visible only in specific pH ranges.

perature:

Fluorescence intensity tends to increase with fall in temperature and to

se to zero at high temperatures. When temperature rises, the motion of

les increases and there is greater te

result in the loss of

fluorescence. With most compounds, a change at 1oC may cause an intensity

change of about 1% [20, 22, 26].

f) Irradiation effect:

The stability of compound when it is radiated by ultraviolet light is an

important consideration in fluorimetry. The extent of photo-decomposition

depends upon the intensity of the light source and as a very intense light source

may enhance the sensitivity of an instrument, it may at the

increased photo-

does occurs, rapid measurements can be carried out before much of the

compound is decomposed. It should be noted that the photo-decomposition

does not always lead to loss of fluorescence and in some cases it leads to the

enhancement of fluorescence [18, 26, 27].

1.6 Experimental observables:

Fluorescent molecules hav

spectrum and the em

18

Chapter 1

am many vibrational and rotational modes. Consequently, their

fluorescence properties are distinctly different from those of small molecules.

Following experimental observables are used to measure the properties

of any luminescent system.

1.6.1 Absorption spectrum:

It shows the dependence of the degree of light absorption by the

compound on the wavelength of light. The quantization condition for the

absorption or emission of light by an atom or by molecule is given by Einstein

relation as given by equation (1.2)

ong

12 EEhc−= h =υ

λ

where E2 and E1 are the electronic energy levels.

The absorption of energy by a molecule is governed by the Beer-Lambert’s law.

According to this relationship,

lcI

..log 010 ε=⎟⎞⎜⎛ (1.4)

I ⎠⎝

where I0 – intensity of incident light,

ted light, I – intensity of transmit

ε – molecular extinction coefficient,

c – concentration of the path length,

l – path length of the absorbing system through which light passes,

and ⎟⎞

⎜⎛

II 0

10log - optical density or ab⎠⎝

sorbance of the material.

In general, the absorption spectrum s plotted in terms of molecular

avelength. The probability of

ound state S00 and the wave function of the

vibrational level of the first excited singlet state S →S .

The positions of the absorption peaks

ution, the broad absorption band is an

indicat cules in the ground state while the

i

extinction coefficient (ε) against frequency or w

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

lowest vibrational level of gr

10 1n

and its nature are of significance

in the spectroscopic studies. In sol

ion of dimeric nature of mole

19

Chapter 1

structu of monomolecular species[28]. But

in soli tructured as in solution. The nature

of abs ular

under study.

1.6.2 Emission Spectrum:

ission spectra. It is observed that the absorption spectra

onal levels of the excited state and the emission

spectra

est vibrational level of S1. This relaxation occur

in abou

y independent of the excitation wavelength [29].

1.6.3 E

red spectrum indicates the existence

ds the absorption spectra are not as s

orption band also give an idea about the lattice structures of molec

system

Emission spectrum defines the relative intensity of radiation emitted at

various wavelengths [18]. The emitted light comprises fluorescence, delayed

fluorescence and phosphorescence, thereby yielding three types of emission

spectra. The Fluorescence emission spectrum shows almost mirror like

symmetry with its absorption.

The delayed fluorescence is specially identical with prompt fluorescence

while the phosphorescence spectrum, although similar in shape is red shifted as

a whole.

The fluorescence emission spectrum is obtained by irradiating the

sample by a wavelength of maximum absorption as given by absorption

spectrum of the sample. The ground state and excited state are associated with

the absorption and em

gives data about the vibrati

yield data about the vibrational levels of the ground state.

The same fluorescence emission spectrum is generally observed

irrespective of the excitation wavelength. Upon excitation into higher

electronic and vibrational levels, the excess energy is quickly dissipated,

leaving the molecule in the low

t 10-12 sec and is presumable a result of strong overlap among numerous

states of nearly equal energy. Because of this rapid relaxation, emission spectra

are usuall

xcitation spectrum:

It defines as the relative efficiency of different wavelengths of exciting

radiation to induce fluorescence. The excitation spectrum is obtained by

measuring the fluorescence intensity at a fixed emission wavelength while the

20

Chapter 1

excitation wavelength is scanned. For large, complex molecules, the excitation

spectrum is quite stable, independent of the emission wavelength.

The excitation spectrum will be identical to the absorption spectrum

where ε.c.l << 1. The measurement of quantum intensity is limited by the

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

excitation source. Parker (1968) 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 [30].

Excitation spectroscopy is also used to determine the quantum efficiency of

r and acceptor molecules.

1.6.4 M

e takes place

almost

energy transfer between dono

irror symmetry:

Mirror image symmetry exists between the absorption or excitation

spectrum and the fluorescence emission spectrum as shown in Figure1.4. The

red side of the absorption and excitation spectrum forms a mirror image of the

blue side of the fluorescence spectrum. This is because fluorescenc

exclusively from the lowest vibrational level of the excited state.

Consequently, the absorption spectrum reflects the vibrational levels of excited

states while the emission spectrum reflects those of the ground state. The

mirror symmetry occurs because the vibrational structures seen in the

absorption and emission spectra are similar, as the spacing of the vibrational

energy levels is not significantly altered by the excitation. The absence of

mirror symmetry indicates a strong interaction in the excited state. For example,

excimers have no mirror symmetry [18].

1.6.5 Stokes shift:

Fluorescence radiation always occurs at wavelengths longer than the

exciting wavelength by a wavelength interval depending on the energy loss in

the excited state due to vibrational relaxation. This phenomenon was first

observed by Stokes in 1852 [5]. This separation between the excitation and

21

Chapter 1

Figure 1.4: Characteristic mirror symmetry of excitation and emission

spectra.

Figure 1.5 : Stokes shift.

emission band maxima is known as the Stokes shift as depicted in Figure 1.5. It

is the characteristic of all complex molecules and usually greater than 10 nm.

22

Chapter 1

When the emission band lies within 30 to 50 nm of excitation wavelength,

measurement problems can arise due to difficulty in separating the Rayleigh

scatter of the excitation light from the emission band. The interactions of solu

molecules with the solvent usually introduce large spectral red shifts of

fluorescence. These shifts are occasionally solvent specific and are also called

Stokes shift [18]. The Stokes shift is of interest to analytical chemists since the

emission wavelength can be greatly shifted by varying the form of the molecule

being excited.

1.6.6 Fluorescence quantum yield:

The quantum efficiency Φ denotes the ratio of the total energy emitted

by any molecule per quantum of energy absorbed. Higher the value of

greater the fluorescence observed of a compound. A nonfluorescent molecule is

one whose quantum efficiency is zero or close to zero that the fluorescence is

te

Φ,

rgy absorbed by such a molecule is rapidly lost by not measurable i.e. all ene

collisional deactivation.

The value of Φ can be determined by measuring the fluorescence of

dilute solution of a standard, such as quinine sulphate, whose quantum

efficiency is known. The fluorescence of the new compound is then measured

and the quantum efficiency is calculated as follows-

unk

std

std

unkstdunknown A

AFF .

.. ×Φ=Φ (1.5)

where F is the relative fluorescence determined by integrating the area beneath

the corrected florescence spectrum, Φ is the respective quantum yields and A is

the absorbance. Quantum yield is characteristic for each fluorescent compound

nd emission wavelengths [31].

1.6.7 F

I and the lifetime τ is

and is independent of the excitation a

luorescence lifetime:

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

23

Chapter 1

I = I0 e-t /τ (1.6)

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 observe

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

ective than

e includes two wavelengths in the form of

excitat

us sample constituents.

The information regarding excitati

ng the excitation and emission

mono

d bandwidths. When working with a mixture

of fluorescent components, t s to

extent of spectral overlaps.

1.7 Flu

fluorescence.

d lifetime is important since it can

Фo ,if one or the other is known.

τ = Фo τo (1.7)

1.6.8 Synchronous fluorescence spectrum:

The fluorescence and phosphorescence methods are more sel

absorptiometry because thes

ion and emission wavelengths. However, conventional emission scans at

fixed excitation wavelength or excitation scan at fixed emission wavelength do

not fully utilize this advantage. These scans provide useful analytical

selectivity only if there are substantial differences in the absorption and/or

luminescence spectral characteristics of the vario

on and emission spectra can be used

more efficiently if synchronous scanning techniques are used. It is possible to

scan the excitation and emission monochromators simultaneously. Often,

synchronous fluorimetry is carried out by scanni

chromators at the same rate while keeping the wavelength difference

between them constant. The main purpose of synchronous scanning is to

generate spectra having decrease

he synchronous scanning is more advantageou

greatly simplify the spectrum and decrease the

orescence quenching:

The lowering of fluorescence intensity by a competing deactivating

process resulting from the specific interaction between fluorophor and another

substance present in the system is called fluorescence quenching [32]. When

one compound diminishes the fluorescence of another, it is said to quench the

24

Chapter 1

A variety of interactions can result in quenching which include excited

state reactions, molecular rearrangements, energy transfer, ground state

hers.

nching, concentration quenching are the other

main t

Dep

n the fluorophore and quencher.

complex formation and collisional quenching.

1.7.1 Quenchers of fluorescence:

A number of substances act as quenchers of fluorescence. One of the

well known collisional quencher is molecular oxygen [33], which quenches

almost all known fluorophors. Therefore it is frequently necessary to remove

dissolved oxygen to obtain reliable fluorescence yields. Aromatic and aliphatic

amines are also efficient quenchers of most unsubstituted aromatic

hydrocarbons [34]. Heavy atoms such as iodide and bromide can also act as

quenchers. The examples stated above are some of the impurity quenc

Besides these, temperature que

ypes of quenching. In temperature quenching, usually with rise in 10C in

temperature, the intensity decreases by 1%. The concentration quenching

includes the decrease in fluorescence intensity with increase in concentration of

fluorescent solution. For this reason in fluorimetric analysis, dilute solutions

are preferred.

ending upon the mechanism of quenching there are two types:

1) Collisional quenching and 2) Static quenching.

Both of these, require molecular contact betwee

1.7.1.1 Collisional quenching:

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.

Collisional quenching of fluorescence is described by the Stern-Volmer

equation-

[ ] [ ]QKQkFF

Dq +=+= 11 00 τ (1.8)

25

Chapter 1

In this equation F0 and F are the fluorescence intensities in the absence and

presence of quencher respectively, qk is the bimolecular quenching constant, τ0

is the lifetime of fluorophor in abs of quencher andence [ ]Q is the concentration

kq τ0. If

the olmer constant will be

of quencher. The Stern-Volmer quenching constant is given by KD =

quenching is known to be dynamic, the Stern-V

represented by KD, otherwise this constant will be described by KSV.

The quenching data are usually presented as plot of F

0 versus [ ]Q

which is known as Stern-Volmer plot.

F

FF0 is expected to be linearly dependent

upon the concentration of quencher. A plot of FF0 versus [ ]Q yields an intercept

of one -1

oncentration at which

on the Y-axis and a slope equal to KD. It is observed that KD is the

quencher cFF0 = 2 or 50% of the intensity is quenche

linear Stern-Volmer plot is generally indicative

The quenching resulting from ground state complex formation between

rns to the

ground

For static quenching, the dependence of the fluorescence intensity upon

ed

constant for complex formation. This constant is given by,

d. A

of a single class of fluorophors,

all equally accessible to quencher.

1.7.1.2 Static quenching:

fluorophor and quencher is called static quenching. The static quenching

provides valuable data regarding the binding between these two molecules.

The ground state complex formed in static quenching process is non-

fluorescent. When this complex absorbs light, it immediately retu

state without emission of a photon.

quencher concentration is easily deriv by consideration of the association

[ ][ ][ ]QF

QFS .

K −= (1.9)

where [F-Q] is the concentration of the complex, [F] is the concentration of

Q] is the concentrati

complexed species are non-fluorescent then the fraction of the fluorescence that

uncomplexed fluorophore, and [ on of quencher. If the

26

Chapter 1

remains (F/Fo) is given by the fraction of the total fluorophores that are not

complexed.

The total concentration of fluorophore [ ] 0.F is given by

[ ] [ ] [ ]QFFF −+=0. (1.10)

by substituting equation ( 1.10) into equation (1.9) we get,

[ ] [ ] [ ][ ][ ] [ ][ ] [ ]QQFQF

K S ..−== (1.11)

We can substitute the fluorophore concentration for fluorescence intensities

and by rearranging equation (1.11)

FFF 10.0 −

[ ]QKFF

S+=0

The dependence of

1 (1.12)

FF0 on [ ]Q is linear as the case in dynamic quenching.

1.7.1.3 Comparison of static and dyna

crease in temperature, the stability of complexes decreases and

conseq

.8 Fluorescence spectrometry:

The fluorescence and fluorescence excitation spectra of the solutions of

arious drug samples as donor and acceptor m

1.6. It has following specifications:

mic quenching:

Dynamic and static quenching constants have different dependency on

temperature. Dynamic quenching depends upon diffusion. With rise in

temperature, diffusion coefficients increases and hence bimolecular quenching

constants are expected to increase with increasing temperature. In contrast to

this, with in

uently it decreases the static quenching constants.

1

1.8.1 Instrumentation:

v olecules in different solvents

were recorded on P. C. based spectrofluorimeter. The experimental set up is

shown in the photograph i.e. Figure

27

Chapter 1

During recording the fluorescence and fluorescence excitation spectra

the parameters like spectral bandwidth (10 nm), data pitch (1 nm) and

wavelength scanning speed (250 nm/min) were kept constant. The other

parameters such as excitation wavelength, emission wavelength were varied as

per the requirement of the experiment.

1.8.2 Optical system of FP-750 spectrofluorimeter:

The optical system of the instrument is given in Figure 1.7. The light

from the source (Xenon lamp) is focused on the entrance slit of the excitation

monochromator by the ellipsoidal mirror M1 and spherical mirror M0. The light

Holographic grating with 1200 lines/mm

: 220 nm to 730 nm

Spectr

avelength scanning speed : 60, 250, 1000, 4000 nm/min

esponse : Fast, Medium, Slow, Auto

ensitivity : Signal to noise ratio of Raman band of

water is higher than 300:1

hotometric display : -999 to +999

ample chamber : Single cell holder (standard)

Detector : Silicon photodiode for Ex.

monochromator and Photomultiplier tube

for Em. monochromator

Instrument : PC based spectrofluorophotometer

Make : JASCO, Japan

Model : FP-750

Light source : 150 W xenon lamp with shielded

lamphouse

Monochromator :

Wavelength range

al bandwidth : 10, 20 nm on both Ex. and Em

monochromator

Wavelength accuracy : ±3 nm

Wavelength threw speed : 30,000 nm/min

W

R

S

P

S

28

Chapter 1

from the slit is dispersed by the diffraction grating G1 and monochromatic light

by the exit slit. A a d to the

ring silicon photodiode, P of splitter, BS, while the

omatic light that has transmitted the beam splitter is led to the sample

e plane mirror M an s focused

on the centre of the sample cell. Th focused on to

f the emission o 4

ors M5 and M . am is taken out from the

he diffractio

after going through the exit slit an etric photomultiplier tube

irror M7

is taken out p rt of the monochromatic light is le

monito S , by the beam

monochr

chamber by th 2 d ellipsoidal mirror M3 where it i

e emission from the sample is

the entrance slit o m nochromator (Em) by ellipsoidal mirror M

and two plane mirr 6 Monochromatic be

light dispersed by t n grating G2 of the emission monochromator

d is led to photom

PMT by the spherical m .

29

Chapter 1

30

Figure 1.6: The experimental setup of the fluorimeter

Chapter 1

Figure 1.7: Optical system of FP-750 Spectrofluorimeter

31

Chapter 1

1.8.3 Detecting and recording system:

The schematic diagram for the FP-750 system is shown in Figure1.8.

The light incident on the monitoring detector (silicon photodiode) and the

emission detector (PMT) is converted into an electrical signal and then

converted into a digital signal by the A/D converter and is introduced to the

microcomputer. The signal subjected to arithmetic operation by the

microcomputer is outputted to the display unit as digital data or spectrum. Both

wavelength as well as slit drives were controlled by the microcomputer.

1.8.4 Operating procedure:

The steps involved during recording of fluorescence and fluorescence

excitation spectra of samples under study are explained as follows-

i) Visual fluorescence color was observed by exciting the sample at

365nm (Hg line) excitation wavelength.

ii) The emission monochromator was set at the approximate wavelength

of visually observed color.

iii) The excitation monochormator was scanned from 250 nm to a

wavelength of emission monochromator.

iv) The excitation spectrum was recorded and the λex was noted.

v) The excitation monochromator was set at λex observed in excitation

spectrum.

vi) The emission monochromator was allowed to scan in the range 300

nm to 750 nm.

vii) The fluorescence emission spectrum was recorded and the λem was

noted.

viii d excitation

spectrum monochromator was scanned and thus the excitation

spectrum is recorded

ix) Finally the fluorescence spectrum was obtained by setting the

excitation monochromator at λex obtained in step (viii). Similarly

fluorescence excitation spectrum was obtained by setting λem

observed in the final emission spectrum.

) The emission monochromator was then set at the λem an

32

Chapter 1

Figure 1.8

: System diagram

33

Chapter 1

1.8.5 Characteristics of an ideal spectrofluorimeter:

To achieve correct analysis by spectrofluorimetric method, the

components of instrument must posses following characteristics-

1.8.5.1: The light source must yield a constant radiation output at all

wavelengths. At present the most versatile light sources are the high pressure

xenon arc lamps. These lamps provide relatively continuous light output from

270 to 700 nm. In addition, the operation of these lamps does not generate

ozone in the surroundings. Xe lamp have useful life of about 2000 hrs. The

lamphouse provides more safety to lamp and analyzer. The high pressure

mercury lamps have higher intensities than Xe lamps, but the intensity is

concentrated in lines. Mercury lamps are only useful if the Hg lines are at

suitable wavelengths for excitation of the fluorophore. Xe-Hg arc lamps, low

pressure Hg lamps are other examples of light sources but these are less

superior than Xe arc lamps.

1.8.5.2: The monochromator must pass radiation of all wavelengths with equal

efficiency. In most of the spectrofluorimeters the diffraction gratings are used

as monochromators. The performance of a monochromator depends on the

dispersion and the straylight levels. For best results, low stray light levels are

required to avoid problems due to scattered stray light. The grating

monochromators may have planar or concave gratings. Planar gratings are

mechanically produced and may contain imperfections while concave gratings

are usually produced by holographic and photo resist methods and having less

imperfections. Imperfections of the gratings are the major source of stray light

transmission by the monochromators and of ghost images from the grating. For

is reason, the holographic gratings are preferable.

.8.5.3: The monochromator efficiency must be independent of polarization.

f monochromator is dependent upon orientation

f polarizer either vertical or horizontal. The polarization characteristics of

onochromators have important consequences in the measurement of

uorescence. Such measurements must be corrected for the varying efficiencies

f each component.

th

1

The transmission efficiency o

o

m

fl

o

34

Chapter 1

1.8.5.4: The detector must detect radiations of all wavelengths with equal

rtional to the

- light absorption by donor.

hoton by the acceptor. The

efficiency. Almost all fluorimeters use photomultiplier tubes (PMT) as

detectors. It is best regarded as a source of current, which is propo

light intensity.

1.9 Energy transfer phenomenon:

1.9.1 Electronic energy transfer mechanism:

The electronic energy mechanism has become one of the most useful

processes in photochemistry having wide applications as a mechanistic tool and

in photochemical synthesis. It allows photosensitization of physical and

chemical changes in the acceptor molecule by the electronically excited donor

molecule. There are two types of energy transfer mechanism-

1.9.1.1 Non-radiative energy transfer:

The process can be defined by the following two steps-

D + hυ → D*

D* + A → D + A* - energy transfer from donor to acceptor.

The electronically excited donor D* is formed initially by direct light

absorption. This can transfer the electronic energy to a suitable acceptor

molecule A resulting in simultaneous quenching of D* and electronic

excitation of A to A*. The transfer occurs before D* is able to radiate and hence

is known as non-radiative transfer of excitation energy. The A* molecule thus

excited indirectly can undergo various photochemical and photophysical

processes. Such processes are called photosensitized reactions.

1.9.1.2 Radiative energy transfer:

The radiative energy transfer involves the trivial process of emission by

the donor and subsequent absorption of the emitted p

process takes place as-

D* → D + hυ

A + hυ → A*

It is called trivial because it does not require any energetic interaction

between the donor and the acceptor. It is merely reabsorption of the

35

Chapter 1

fluorescence radiation. Though it is called trivial, it causes radiation

imprisonment and may introduce error in fluorescence measurement [11].

1.9.2 Förster (Fluorescence) Resonance Energy Transfer (FRET):

It is a physical phenomenon described over 50 years ago, that is being

l research and drug discovery today. It

r mechanism between two

chrom

rst paper

portance of energy in photosynthesis.

sonance energy

a)

e intensity and excited state lifetime

and increase in the acceptor’s emission

bove, there should be proximity between donor and

acceptor (10-100 Å) and absorption/ excitation of acceptor must overlap with

emission spectrum n in figure 1.9.

used more and more in biomedica

describes a non radiative energy transfe

ophores. It is generally acronymed as FRET.

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. By 1946, Professor Förster had written his fi

on energy transfer and pointed out the im

When both molecules are fluorescent, the term fluorescence re

transfer is used.

The efficiency of FRET depends on the following parameters-

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 fluorescenc

intensity. A pair of molecules that

interacts in such manner that FRET occurs is referred as donor-acceptor pair.

As mentioned a

of the donor as show

36

Chapter 1

with emission of

1.9.3 C

rster

dis 0 nge of 20 to 60 Å [35].

The ra

Figure 1.9: Spectral overlap of excitation of acceptor

donor.

haracteristics of FRET:

The distance at which energy transfer is 50 % efficient is called Fö

tance (R ), which is typically in the ra

te of energy transfer from a donor to an acceptor is given by-

( )6

01⎟⎠⎞

⎜⎝⎛=

rR

rkD

T τ (1.13)

where Dτ is the decay time of the donor in the absence of acceptor, R0 is the

Förster distance and r is the donor to acceptor distance. Hence the rate of

transfer is equal to the decay rate of the donor ⎟⎟⎠

⎞⎜⎜⎝

⎛

Dτ1 when the D-A distance (r)

is equal to the Förster distance (R0) and the transfer efficiency is 50 %. At

37

Chapter 1

r = R0, the donor emission would be decreased to half its intensity in the

absence of acceptor. Thus the rate of FRET depends strongly on distance and is

proportional to r -6 [29].

Förster distances in the range 20-90 Å are convenient for studies of

biological macromolecules, vitamins, drug molecules etc. Any condition that

affects the D-A distance will affect transfer rate, allowing the change in

distance to be quantified. In such applications, the extent of energy transfer

between a fixed donor and acceptor is utilized to calculate the D-A distance

and thus to obtain structural information about the molecule. Such distance

measurement is important aim of FRET and hence it is called as ‘spectroscopic

ruler’ [36, 37]. The use of the energy transfer as a proximity indicator

illustrates an important characteristic of energy transfer. It is observed that

FRET will occur if the spectral properties are suitable and the D-A distance is

comparable to R0. A wide variety of biochemical interactions results in changes

in distance and thus can be calculated using FRET.

1.9.

1.9. r of energy:

energy transfer have been derived from

4 Theoretical aspects of energy transfer:

4.1 Rate of transfe

The theory of resonance

classical and quantum mechanical considerations. The rate of transfer for a

donor and acceptor separated by distance r is given by [29]-

( ) ( ) ( ) ( ) λλλελ dFKrk ADD

T4

456

2 10ln9000∫πτ NnrD 0128

∞

⎠⎝

N = Avogadro’s number

⎟⎞

⎜⎛Φ

= (1.14)

where DΦ = the quantum yield of the donor in absence of acceptor,

n = refractive index of medium,

r = is the distance between the donor and acceptor ,

Dτ = is the lifetime of donor in absence of acceptor ,

( )λDF is the corrected fluorescence intensity of the donor in the wavelength

range λ to λ + ∆λ with the total intensity (area under curve) normalized to unity,

38

Chapter 1

( )λε A is the extinction coefficient of the acceptor at λ. The term K2 is a factor

describing the relative orientation in space of the transition dipoles of the donor

and acceptor. K2 is usually assumed to be equal to 2/3 which is appropriate for

dynamic random averaging of the donor and acceptor.

The overlap integral (J) expresses the degree of spectral overlap

between the donor emission and the acceptor absorption.

( ) ( )( ) ( )

( )∫

∫∫ ∞

∞

∞

==

0

0

4

0

4

λλ

λλλελλλλελ

dF

dFdFJ

D

AD

AD (1.15)

F

D (λ) is dimensionless. If ( )λε A is expressed in units of M-1cm-1 and λ is in

nm, then J is in units of M-1cm-1 (nm)4.

1.9.4.2 Förster distance (Ro):

For biochemical processes it is usually convenient to consider distances

than transfer rates. For this reason equation 1.14 is written in terms of the

ecules decay by energy transfer

with

Förster distance R0 at which half the donor mol

( ) 1−= DT rk τ ,following equation is from equations (1.13) and (1.14)

obtained [29].

( ) ( ) ( ) λλλελπ

dFnNKR AD

D 4

045

26

0 .12810ln.9000

∫∞

expression, Förster distance can be calculated from the spectral

properties of the donor and acceptor and the donor quantum yield.

This expression can be made simpler to calculate R0 in terms of the

g the

ngth is expressed in nm then

Φ= (1.16)

From this

experimentally known values which is accomplished by combinin

constant terms in equation (1.16). If the wavele

( )λDF is in units of M-1cm-1(nm)4 and the Förster distance in Å is given by –

( ) 6/1420 211.0 JnKR DΦ= − (1.17)

( )JnKR DΦ×=∴ −− 4256 1079.8 (1.18) 0

length is expressed in cm and J is in units of M-1cm3 then,

If the wave

( )JnKR −− 42256 1079.8 (in cm6) (1.19) DΦ×=0

39

Chapter 1

The

the transfer rate is much

faster t

otons absorbed by the

donor which are transferred to the acceptor [29]. The fraction is given by

rate of energy transfer can be easily calculated by knowing R0 from

equation (1.13).

1.9.4.3 Efficiency of energy transfer:

The energy transfer will be efficient only when

han the decay rate. If reverse is the case then FRET will be inefficient.

The efficiency of energy transfer (E) is the fraction of ph

( )( )rk

rkETD

T

+= −1τ

which is the ratio of the transfer rate to the total decay rate of the donor in the

(1.20)

presence of acceptor. By substituting equation (1.13), equation (1.20) can be

rearranged as

660 rR

E+

= (1.21)

From this equation it is clear that the efficiency of the energy transfer is

strongly dependent on distance when the D-A distance is near R

60R

nsfer efficiency is typically measured using the relative

fluorescence intensity of the donor, in the absence (F0) and presence (F) of

acceptor.

0. The

efficiency quickly increases to 1 as the D-A distance decreases below R0.

The tra

0

1FFE −= (1.22)

1.9.4.4

that o

yield of the donor

Various terms involved in calculations:

To calculate the D-A distance, it is necessary to know R0, which in turn

depends upon K2, n, DΦ and J. The refractive index is often assumed to be near

f water (n =1.33) or small organic molecules (n =1.39). The quantum

DΦ is determined by comparis

The overlap integral must be evaluated for each D-A pair. The greater the

overlap of the emission spectrum of the donor with the

tinction

coefficients result in larger R0 values .The orie 2 is nt

on with standard fluorophors.

absorption spectrum of

the acceptor, the higher the value of R0. Acceptors with larger ex

ntation factor K depende

40

Chapter 1

upon geometrical considerations of emission transition dipole of the donor and

the absorption transition dipole of the acceptor. It is generally assumed equal to

acceptors that randomize by rotational

mon applicati

between two sites on a macromolecules.

d to study macromolecular system when there is more than a

ule near a d

d) It can be used to measure binding interactions between molecules in

these

acellular probes and for making the surfaces functional. FRET can be

such inte

f) FRET has been used to develop a number of sensors. The use of donor

because the measurements

2/3, which is the value for donors and

diffusion prior to energy transfer [29].

1.9.4.5 Applications of FRET:

FRET has number of applications in various areas of science. Some of

these are summarized as follows:

a) The most com on of FRET is to measure the distances

b) It can be used to measure the extent of binding. The steady state

measurements are often used to measure binding interactions.

c) It is use

single acceptor molec onor molecule.

solution [38] or in microscopy [39-40].

e) Quantum dots [QDs], semiconductor nanoparticles have high quantum

yields, narrow emission spectra and good photostability and hence

are widely used as fluorescent probes. The knowledge of the

interactions of QDs with proteins is important for their use as

intr

used in understanding ractions [41].

to acceptor intensity ratios is valuable

become mostly independent of the overall intensity. This independence

is important in fluorescence microscopy where it is not possible to

control the local fluorophore concentration. This property has been used

to develop sensors for a variety of analytes [42].

g) FRET is used extensively in DNA analysis. The donor-acceptor pairs

can be designed which display distinct emission spectra and similar

intensities with a single excitation wavelength. Such pairs with their

FRET mechanism can be used for DNA sequencing [43,44].

41

Chapter 1

h) FRET is to be used with fluorescence life time imaging microscopy

(FLIM) to study association reactions in cells [45-46].

1.10 Binding Mechanism:

When the interaction occurs between two different molecules, the

constant and number of binding sites can be

determi

binding parameters like binding

ned by following equation [47].

[ ]QnKF

F

FFloglog 0 =

−log+ (1.23)

where ssed earlier, K is the binding

con

interac

easily

0 and F have same meaning as discu

stant, n is the number of binding sites for that particular molecular

tion and [Q] is the concentration of quencher.

The binding constant (K) and the number of binding sites (n) can be

determined by plotting the graph of F

FF −0log versus [ ]Qlog . The nature

of this plot will be a straight lin Y-axis. The slope

determ

constant of that interaction.

1.11 Thermodynamic parameters:

(ΔS) and the enthalpy change (

fluorescenc o

determ

quenchi

recorded at various temperatures by

binding constants are related with

equation,

e with intercept on

ines the number of binding sites while intercept gains the binding

One can determine the change in free energy (ΔG), the entropy change

ΔH) for any particular interaction by using

e quenching data. By applying equation 1.23, it is possible t

ine binding constants at various temperatures, with fluorescence

ng measurements. The fluorescence of the system under study can be

keeping it in thermostat. The various

different temperatures by van’t Hoff

RS

RTHK Δ

+Δ

−=ln (1.24)

temper

accom

here, K is binding constant, R is the gas constant, T is absolute

ature, ΔH is change in enthalpy and ΔS is change in entropy

panying the molecular interaction.

42

Chapter 1

obtain

G = ΔH –TΔS (1.25)

n the different molecules essentially

includes, hydrogen bond, van der Waals’ force

hydrophobic interaction [48]. Ross summed up the thermodynamic laws for

force while if both are

ion mainl py dr

ΔH ≈ 0 and ΔS have positive value then there will be electrostatic force

sfer was introduced and developed

ET [54].

Förster calculated the critical distanc

hodamine B in glycerol,2-5 diphenyl

oxazole, 9-10 dibromoanthracene in cyclohexane, etc [55]. Förster himself

Subsequently, free energy change at different temperatures can be

ed by Gibb, s equation,

Δ

Thus, fluorescence study enables to get idea about thermodynamic

parameters. The nature of binding forces can be predicted by observing these

parameters. The acting forces betwee

s, electrostatic interactions and

estimating the type of the binding force between organic micromolecule and

biological macromolecule. If ΔH and ΔS are both positive or having some

higher values the main force will be hydrophobic

negative or some lower values then hydrogen bond and van der Waals’ forces

will be key forces of interaction and react y enthal iven. Also if

between the acting molecules. The negative value of ΔG will indicate

spontaneity of reaction and vice-versa[49-52].

1.12 Literature survey on FRET studies:

The concept of resonance energy tran

by Professor Theodor Förster. J.Perrin was the first person to realize the

interaction from one molecule to another through interactions between

oscillating dipoles of closely placed molecules [53]. Förster proposed a non-

radiative mechanism for FRET which includes long range dipole-dipole

interactions and energy transfer between donor and acceptor which arises from

mutual resonance dipole perturbation. He explained the energy transfer

phenomenon in photosynthesis in his first paper and developed a correct

theoretical basis of FR

e between donor and acceptor at

which the efficiency of energy transfer will be 50 % which is called as Förster

distance [35]. A.Kawasaki et al confirmed the Förster’s theory of RET with

various donor-acceptor pairs containing r

43

Chapter 1

confirmed the validity of energy transfer theory with fluorescein and

rhodamine B in glycerol and with fluorescein and rhodamine 6G [56].

J.Hung et al [57] st

-diethylaniline.

he interaction of BSA with 6-

mercap

ct was utilized by L.Matyus et al [63].The

interac

udied the interaction between antileishmanial drug,

Boldine, with the promastigotes of leishmania mexicana and applied the FRET

to determine the mechanism of biological action of the drug. Y.Li et al [58]

applied FRET for studying the molecular interaction between dibucaine and

acriflavine in aqueous as well as in polystyrene latex dispersions. Dibucaine is

local anesthetic drug which was donor and acriflavine, having role of sensitizer

in photodynamic therapy, was acceptor molecule. The studies shown that the

molecular interaction involved electrostatic and hydrophobic forces.

The binding of antimicrobial drug, Gatifloxacin with BSA molecule was

studied by M.Guo et al [59] by using fluorescence quenching mechanism. The

binding parameters as well as effect of drug on conformation of BSA has been

analysed by synchronous fluorescence spectroscopy. M.H.Zhang et al [60]

reported the fluorescence study between Chinese herbal medicine Hypocrellin,

having activities against skin diseases, and N-N

Y.J. Hu et al [61] investigated t

topurine (6-MP), an antiviral agent of purine series. The change in

conformation of BSA in presence of 6-MP, dynamic quenching of BSA

fluorescence by 6-MP and thermodynamic parameters were studied. S.

Chatterjee et al [62] observed the interaction between anionic dye fluorescein

and cationic dye safranine T in aqueous as well as micellar medium which

includes the effect of viscosity on the FRET by fluorescence anisotropy study.

Fluorescence quenching studies used to obtain adequate information

about the structure and dynamics of biologically important macromolecular

systems like proteins. This fa

tion between well known dye methylene blue and BSA was studied by

Y.J.Hu et al [64] with respect to FRET and all corresponding parameters. The

fluorescence quenching of HSA by antidepressant drug doxepine hydrochloride

involves static quenching mechanism was concluded by P.B.Kandagal et al

[65]. The study included determination of intermolecular distance between

44

Chapter 1

donor and acceptor, critical distance, thermodynamic parameters of binding

process, etc. Further more it pointed at the possible binding site between

protein

studied by

X.Che

nor. From this

interac

,

interfe

and drug.

C.Wang et al [66] studied the interaction of carbamazepine with BSA

and determined the binding, thermodynamic parameters. The fluorescence

quenching study between riboflavin and serum albumins was carried out by

Z.Hongwei et al [67]. The binding constants and binding sites were obtained at

various temperatures.

An antibiotic ,lomefloxacin is a drug belonging to fluoroquinolones. The

energy transfer between bovine lactoferrin and lomefloxacin was

n et al [68].The change in conformation of bovine lactoferrin is

determined by this study. The binding mechanism of anti inflammatory drug

cromolyn sodium to BSA was investigated by Y.Liu et al [69] with respect to

nature and magnitude of interaction.

The flavonoid nevadensin acts as quencher of lysozyme fluorescence

intensity. Lysozyme is small monomeric protein acts as do

tion, critical distance of energy transfer, the distance between donor and

acceptor, etc were calculated by D.Li et al [70].An alkaloid class drug brucine

interacted with HSA which was investigated by Y.Q.Wang et al [71]

spectrofluorimetrically from which binding parameters has been calculated.

From this review, we planned to utilize fluorescence properties of some

drug molecules to investigate drug-drug, drug-vitamin, drug-BSA molecular

interactions from which the binding parameters, drug absorption, distribution

rence of coexisting species can be determined.

45

Chapter 1

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49