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Chapter 1. Introduction

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1. Introduction

1.1 Literature Survey and Synthesis of Heterocyclic Compounds:

Heterocyclic compounds particularly five or six membered ring compounds

have occupied the first place among various classes of organic compounds for

their biological and pharmacological activities. As pyrimidine is a basic nucleus in

DNA and RNA, it has been found to be associated with diverse biological

activities [1, 2]. Pyrimidine moiety is an important class of N-containing

heterocycles widely used as key building blocks for pharmaceutical agents. It

exhibits a wide spectrum of pharmacophore as it acts as bactericidal, fungicidal,

analgesic, anti-hypertensive, and anti-tumor agent [3-6].

The chemistry of pyrimidine and its derivatives has been intensively

studied because of the pharmacological and physical properties of these important

heterocycles. Pyrimidine derivatives, including uracil, thymine, cytosine, adenine,

and guanine, are fundamental building blocks of deoxyribonucleic acids (DNA)

and ribonucleic acids (RNA). Vitamin B1 (thiamine) is a well-known example of a

naturally occurring pyrimidine that is encountered in our daily lives. Synthetic

pyrimidine derivatives are used in the pharmaceutical industry as potent drugs. For

example, pyrimethamine is used as an antimalarial and antiprotozoal drug that is

used in combination with sulfadiazine [7-10]. Pyrimidines also play a role as

analgesic, antihypertensive, antipyretic, antiinflammatory, antineoplastic,

antibacterial, antiprotozoal, antifungal, antiviral, and antifolate drugs and as

pesticides, herbicides, and plant growth regulators [11-17]. In recent studies it was

shown that bicyclic pyrimidine nucleosides are potent and selective inhibitors of

Varicella Zoster Virus (VZV) replication [18-20]. Tao Wang and co-workers

reported that pyrimidine derivatives were identified as potent inhibitors of TrK

kinase. The latter plays a critical role in cell signaling and cancer related processes

[21].

Pyrimidine derivatives were intensively investigated as electroluminescent

materials in the past and as a two-photon absorption organic chromophores [22-


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24]. The influence of the pyrimidine additives on the dye sensitized solar cell

performance was also investigated [25]. The effect of solvents on the spectral

properties of molecules, generally referred as solvatochromism, has been

investigated for many years, generating a copious of literature [26]. Recently some

marine alkaloids such as dihydropyrimidine-5-carboxylate have been synthesized

and used as fluorescent probes. They exhibit interesting biological activities like

potent HIV-gp-120-CD 4 inhibitors as well as anti-HIV agents [27, 28].

Fluorescent probe has been widely used in the field of biological and organic

material science. Some pyrimidine derivatives are fluorescent materials that

possess many valuable photophysical properties. In recent years considerable

efforts have been made to the design and synthesis of functional molecules that

could serves as sensitive sensors for the analytical detection of chemically and

biologically important species [29]. For this purpose, the advantages of

fluorescence signaling in high selectivity and sensitivity have encouraged the

development of a variety of interesting and practically usable fluorescent probes

[30, 31]. Pyrimidines are also of considerable importance in the field of material

sciences. For example, they have been reported [32] to be efficient organic light

emitting devices (OLED), which play an important role in biological and material

sciences [33-42]. Furthermore, some fluorescent chemosensors for detections of

metal ions have been found in literature derived from pyrimidine derivatives [43].

The biological and photophysical significance of the pyrimidine derivatives

has led us to the synthesis of substituted pyrimidines and investigation of their

photophysical characteristics. The development of simple synthetic routes for

widely used organic compounds from readily available reagents is one of the

major tasks in organic synthesis. Nowadays, the one step methods involving three-

component condensation are popular in synthetic organic chemistry for the

synthesis of heterocyclic compounds. The one step methods are more convenient

as compared with multistep, since they require shorter reaction time and gives

higher yield with easy workup. Heterocycles are ubiquitous to among


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pharmaceutical compounds. The biological and photophysical significance places

this scaffold at a prestigious position in medicinal chemistry research.

Scientist P. Biginelli in 1893 reported one-step synthesis of 3, 4-

dihydropyrimidin-2(1H)-one by three-component condensation of aldehydes, ethyl

acetoacetate and urea in alcohol using strong mineral acid. These Biginelli

compounds possess several pharmaceutical properties like anti-bacterial, anti-

viral, anti-inflammatory, anti-hypertensive and anti-tumor agents. The scope of the

original Biginelli reaction was gradually extended by variation of all three

building blocks, allowing access to a large number of multifunctionalized

dihydropyrimidones. Although, various methods are reported concerning the

synthesis of pyrimidine derivatives, few one-pot syntheses [44, 45] have been

published using aromatic aldehydes, ethyl cyanoacetate and thiourea.

In this research work, we have been reporting the synthesis of nitrogen

containing heterocyclic compounds by three-component condensation of aromatic

aldehydes, ethyl cyanoacetate and guanidine hydrochloride in ethanol under

alkaline medium and study on their photophysical behaviour. These pyrimidine

derivatives are synthesized by reported literature method (Scheme 1.1) [46].

HO

R

OC2H5NC

O NH2 NH2

NH NaOH

EtOHReflux

N

NNH2 OH

CN

R

+ + HCl.

Scheme 1.1 Representative synthesis of compounds (a-d).

1.1.1 General procedure for the preparation of desired compounds:

The equimolar mixture of aromatic aldehydes (10 mmol), ethyl cyano

acetate (10 mmol) and NaOH (0.4g in 5 mL water) in 25 mL ethanol was stirred

mechanically for at least 10 minutes, and then guanidine hydrochloride (10 mmol)

was added to the reaction mixture. The above reaction mixture was refluxed till


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the completion of reaction as monitored by TLC. After the completion of reaction,

the reaction mixture was poured into ice-cooled water and neutralized by 1:1 HCl

to get the desired product. The separated solid was filtered, washed with little

distilled water to remove excess of acid. Finally, the crude product was purified by

recrystallisation from ethanol to get pure product (Scheme 1). The purity of the

desired compounds was tested by physical constants and they are used for the

investigation of photophysical behaviour. The data have been reported in Table

1.1

Table 1.1: Synthesis of 2-amino-5-cyano-6-hydroxy-4-aryl pyrimidines (a-d).

Fluorescence quenching studies used to obtain adequate information about

the structure and dynamics of biologically important macromolecular systems like

proteins. The interactions between transporter proteins and various types of

ligands have been investigated for many years. The fluorescence quenching of

Human serum albumin (HSA) by antidepressant drug doxepine hydrochloride,

involving static quenching mechanism was reported by P. B. Kandagal et al [47].

The study included determination of intermolecular distance between donor and

acceptor, critical distance, thermodynamic parameters of binding process, etc.

Furthermore it also pointed out the possible binding site between protein and drug.

C. Wang et al [48] studied the interaction of carbamazepine with Bovine

serum albumin (BSA) and determined the binding, thermodynamic parameters.

The fluorescence quenching study between riboflavin and serum albumins was

Sr. No.(Entry) Aryl aldehydes Time (h) Yield (%)

a C6H5-CH=CH- 1.5 91

b 4-OHC6H4 1.5 92

c 3,4-(OCH3)2 –C6H3 1.5 90

d 4-N,N-(CH3)2-C6H4 1.0 91


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carried out by Z. Hongwei et al [49]. 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 studied by X.

Chen et al [50].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 [51] with respect to nature and magnitude of

interaction.

As the synthesized pyrimidine derivatives exhibits biological activity, from

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

derivatives to investigate drug-protein molecular interactions. The proteins used

for the interaction studies are Human serum albumin (HSA) and Bovine serum

albumin (BSA), from which the binding parameters, drug absorption, distribution,

conformational changes in proteins can be determined. Some pyrimidine

derivatives are also used as a chemosensors for the detection of metal ion and

water composition in binary aqueous solution.

1.2 General Introduction to Photochemistry:

Photochemistry is concerned with the absorption, excitation and emission

of photons by atoms, atomic ions, molecules, molecular ions, etc. Simplest

photochemical process with the absorption and subsequent emission of a photon

by a molecule (A) is shown in scheme 1.2. When the molecule (A) absorbs a

photon it is said to be excited. After a short period of time, the excited state

molecule emits a photon of certain light and falls back to the ground state.

A molecule in the electronically excited state is completely different

chemical species with its own wave function and nuclear geometry. Since the

charge densities are different, it shows a different chemistry from the normal

ground state molecule, because it has excess energy but weaker bonds. The

physical properties such as dipole moment, pK values, and redox potentials differ

in comparison to the ground state values. Excited states, in general, have less deep


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minima in their potential energy surfaces, indicative of the weakening of attractive

interactions. Usually the equilibrium internuclear distances increase and some of

the states may be completely repulsional, leading to direct dissociation on

transition.

Scheme 1.2: Photoexcitation of molecule

1.2.1 Luminescence:

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 incandescence where

luminescence refers to cold light and incandescence refers to hot light.

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 [52,53].

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 1.2.


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Table 1.2: 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

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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

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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

1.2.2 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-


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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 [54,55].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 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 [56].

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 [57].

Nowadays there has been tremendous growth in the use of fluorescence in

various branches of science. Fluorescence spectroscopy and time resolved


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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.

1.2.3 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


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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.

1.2.4 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 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 [58,59]. The Jablonski diagram has many forms; one such

classical diagram is as shown in Figure 1.1.

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 molecules 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


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

those of fluorescence.

Figure 1.1: Jablonski diagram

1.2.5 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 phosphorescence is

long lived delayed emission having spectral characteristic very different from

fluorescence. However, there is delayed emission whose spectrum 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 [60].


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1.2.5.1 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 (11 TSE −∆ ) 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 (S1). 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 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 [61].

1.2.5.2 P-type delayed fluorescence:

If energy gap between singlet and triplet state (∆ES–T) 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) Unimolecular processes:

After absorbing the radiation molecule from ground state goes excited state

as represented:


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A + hv → A*

where A* is either an electronically excited molecule with excess vibrational

energy in S1 state or a molecule excited to higher singlet states S2, S3, etc. The

various photophysical processes that can occur in a molecule are:

Scheme 1.3: Unimolecular process

where A*, 3A, and A are molecules in excited singlet state, molecules in triplet

state and in the ground state respectively. In radiationless or nonradiative

transitions processes such as an internal conversion and intersystem crossing, the

excess energy is lost to the environments as thermal energy. Some of the

unimolecular processes are represented by Jablonski diagram [62, 63].

2) Bimolecular photophysical processes:

The main bimolecular photophysical processes responsible for deexcitation

of molecules are presented in scheme. It is interesting to note that some of them

involve energy transfer, electron transfer, photon transfer, impurity quenching,

solvent quenching, self quenching etc and are represented as in [64,65]


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Scheme 1.4: Bimolecular photophysical processes

In this process, a molecule initially excited by absorption of radiation and

interacts with another molecule by nonradiative mechanism. The second molecule,

thus excited can undergoes various photophysical and photochemical processes

according to its own characteristics. The fluorescence characteristics of A* are

affected by the presence of Q as a result of competition between the intrinsic de-

excitation and intermolecular processes. Solvent quenching may involves other

physical parameter such as solute-solvent interactions. Since the solvent acts as the

medium in which the solute molecules are bathed, solvent quenching may

classified under unimolecular processes and a clear distinction between this

unimolecular processes and internal conversion S1 →S0 is difficult.

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 seem 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 compete with fluorescence.


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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 [66]. 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 transient thermal


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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 [67].

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


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[68]. 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 state has significance in producing delayed

fluorescence and phosphorescence, which is radiative decay of triplet state

molecule to the ground state.

1.5 Characteristics of fluorescence:

The phenomenon of fluorescence has a number of general characteristics.

The fluorescence spectrum recorded is 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:

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

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

F = (I0 – I) ΦF

If ΦF is constant then the shape of the fluorescence spectrum is determined

solely by the extinction coefficient ε of the molecule [69, 70].

1.5.2 Factors influencing the fluorescence intensity:

Fluorescence intensity of a compound is altered by following factors-

a) Structure of the compound:

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

which exhibit fluorescence are usually aromatic or contain conjugated double

bonds. As fluorescence is an electronic phenomenon, the molecules having readily

available electrons for energy transitions are capable to show fluorescence. Such

electrons are π or delocalized electrons and ‘lone-pair’ electrons.

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

show fluorescence and if a substituent, increasing the freedom of these electrons,

is added to the compound then the substituted compound is likely to be more


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fluorescent than the parent compound [70, 71]. On the other hand, if the

substituent tends to localize the π-electrons, there will be a diminution of

fluorescence.

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

more fluorescent than benzene and its derivatives. Naphthalene, anthracene and

biphenyl derivatives [72, 73] 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 of the compound to be assayed is very important consideration in

quantitative work [74]. 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 at high dilutions where the number of

molecules present is small enough to make the extent of re-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:


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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 fluorescence related work.

All solvents should be free from contaminants which may enter

through cleansing agents of glassware. For example, chromic acid absorbs

ultraviolet light and hence it is preferable to clean cuvettes in nitric acid

rather than chromic acid.

ii) Non-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

showed 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 of

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

an increase of phosphate concentration frequently leads to diminution in

fluorescence intensity.

d) pH of the solution:

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

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


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

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

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

fluorescent indicator is based on the pH-fluorescence change. In such indicators,

fluorescence is visible only in specific pH ranges.

e) Temperature:

Fluorescence intensity tends to increase with fall in temperature and to

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

molecules increases and there is greater tendency for collisions. This would result

in the loss of some of the energy which might have radiated as fluorescence. With

most compounds, a change at 1oC may cause an intensity change of about 1% [69,

71, 75].

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 same time cause

increased photo-decomposition. However, even though photo-decomposition does

occur, 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 [67, 75, 76].

1.6 Experimental observables:

Fluorescent molecules have two characteristic spectra: the excitation

spectrum and the emission spectrum. The large molecule have large number of

electrons and nuclei hence the absorbed energy can be readily distributed among

many vibrational and rotational modes. Consequently, their fluorescence

properties are distinctly different from those of small molecules.


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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,

λυ hc

hE == (1.1)

12 EEhc

h −==λ

υ

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

I..log 0

10 ε=

(1.2)

where I0 – intensity of incident light,

I – intensity of transmitted light,

ε – molecular extinction coefficient,

c – concentration of the path length,

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

and

I

I 010log - optical density or absorbance of the material.

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

extinction coefficient (ε) against frequency or wavelength. The probability of the

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

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

of the first excited singlet state S10→ S1n .


22

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

the spectroscopic studies. 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 [77]. But in solids the absorption

spectra are not as structured as in solution. The nature of absorption band also

gives an idea about the lattice structures of molecular system under study.

1.6.2 Emission Spectrum:

Emission spectrum defines the relative intensity of radiation emitted at

various wavelengths [67]. 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

emission spectra. It is observed that the absorption spectra gives data about the

vibrational levels of the excited state and the emission spectra 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 lowest vibrational level of S1. This relaxation occur in about 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 usually

independent of the excitation wavelength [78].

1.6.3 Excitation spectrum:


23

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

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 [79]. Excitation spectroscopy is also

used to determine the quantum efficiency of energy transfer between donor and

acceptor molecules.

1.6.4 Mirror symmetry:

Mirror image symmetry exists between the absorption or excitation

spectrum and the fluorescence emission spectrum as shown in Figure1.2.

Figure 1.2: Characteristic mirror symmetry of excitation and emission

spectra.


24

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

image of the blue side of the fluorescence spectrum. This is because fluorescence

takes place almost 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 [67].

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 [54]. This separation between the excitation and emission band

maxima is known as the Stokes shift as depicted in Figure 1.3.

Figure 1.3: Stokes shift.


25

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

nm. 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 solute

molecules with the solvent usually introduce large spectral red shifts of

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

Stokes shift [67]. 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 Φ, 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 not measurable

i.e. all energy absorbed by such a molecule is rapidly lost by 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

A

F

F .

.. ×Φ=Φ (1.3)

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 and is

independent of the excitation and emission wavelengths [80].


26

1.6.7 Synchronous fluorescence spectrum:

The fluorescence and phosphorescence methods are more selective than

absorptiometry because these include two wavelengths in the form of excitation

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 various sample constituents.

The information regarding excitation 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 scanning the excitation and emission

monochromators at the same rate while keeping the wavelength difference

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

spectra having decreased bandwidths. When working with a mixture of fluorescent

components, the synchronous scanning is more advantageous to greatly simplify

the spectrum and decrease the extent of spectral overlaps.

1.7 Fluorescence 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 [81]. When one compound

diminishes the fluorescence of another, it is said to quench the fluorescence.

A variety of interactions can result in quenching which include excited state

reactions, molecular rearrangements, energy transfer, ground state 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 [82], which quenches almost all


27

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 [83]. Heavy

atoms such as iodide and bromide can also act as quenchers. The examples stated

above are some of the impurity quenchers. Besides these, temperature quenching,

concentration quenching are the other main types 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.

Depending upon the mechanism of quenching there are two types:

1) Collisional quenching and 2) Static quenching.

Both of these require molecular contact between the fluorophore and quencher.

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 fluorophore 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-

[ ] [ ]QKQkF

FDq +=+= 11 0

0 τ (1.4)

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 fluorophore in absence of quencher and [ ]Q is the concentration of

quencher. The Stern-Volmer quenching constant is given by KD = kq τ0. If the


28

quenching is known to be dynamic, the Stern-Volmer constant will be represented

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

The quenching data are usually presented as plot of F

F0 versus [ ]Q which is

known as Stern-Volmer plot. F

F0 is expected to be linearly dependent upon the

concentration of quencher. A plot of F

F0 versus [ ]Q yields an intercept of one on

the Y-axis and a slope equal to KD. It is observed that KD -1 is the quencher

concentration at whichF

F0 = 2 or 50% of the intensity is quenched. A linear Stern-

Volmer plot is generally indicative of a single class of fluorophors, all equally

accessible to quencher.

1.7.1.2 Static quenching:

The quenching resulting from ground state complex formation between

fluorophore 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 returns to the ground

state without emission of a photon.

For static quenching, the dependence of the fluorescence intensity upon

quencher concentration is easily derived by consideration of the association

constant for complex formation. This constant is given by,

[ ][ ][ ]QF

QFKS .

−= (1.5)

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

uncomplexed fluorophore, and [Q] is the concentration of quencher. If the

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


29

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. (1.6)

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

[ ] [ ]

[ ][ ][ ]

[ ][ ] [ ]QQF

F

QF

FFK S

1

..0.0 −=

−= (1.7)

We can substitute the fluorophore concentration for fluorescence intensities and by

rearranging equation (1.7)

[ ]QKF

FS+= 10 (1.8)

The dependence of F

F0 on [ ]Q is linear as the case in dynamic quenching.

1.8 Energy transfer phenomenon:

1.8.1 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.

1.8.1.1 Non-radiative energy transfer:

The process can be defined by the following two steps-

D + hυ → D* - light absorption by donor.

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


30

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.

1.8.1.2 Radiative energy transfer:

The radiative energy transfer involves the trivial process of emission by the

donor and subsequent absorption of the emitted photon by the acceptor. The

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 fluorescence

radiation. Though it is called trivial, it causes radiation imprisonment and may

introduce error in fluorescence measurement [60].

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

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

more and more in biomedical research and drug discovery today. It describes a

non radiative energy transfer mechanism between two chromophores. 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 first paper on energy transfer

and pointed out the importance of energy in photosynthesis. When both molecules

are fluorescent, the term fluorescence resonance energy transfer is used.

The efficiency of FRET depends on the following parameters-


31

a) the distance between donor and acceptor molecules,

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

absorption spectrum of the acceptor,

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

d) the quantum yield of the donor.

Figure 1.4: Spectral overlap of excitation of acceptor with emission of Donor.

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

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

leads to reduction in donor’s fluorescence intensity and excited state lifetime and

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

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

As mentioned above, there should be proximity between donor and

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

emission spectrum of the donor as shown in figure 1.4.


32

1.8.3 Characteristics of FRET:

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

distance (R0), which is typically in the range of 20 to 60 Å [84]. The rate of energy

transfer from a donor to an acceptor is given by

( )6

01

=r

Rrk

DT τ

(1.9)

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

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 [78].

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’ [85, 86]. 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.

1.8.4 Theoretical aspects of energy transfer:


33

1.8.4.1 Rate of transfer of energy:

The theory of resonance energy transfer have been derived from classical

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

acceptor separated by distance r is given by [78].

( ) ( ) ( ) ( ) λλλελπτ

dFNnr

Krk AD

D

DT

4

0456

2

128

10ln9000∫∞

Φ= (1.10)

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

n = refractive index of medium,

N = Avogadro’s number,

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, ( )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. K

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

dF

dFJ

D

AD

AD (1.11)

FD (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.

1.8.4.2 Förster distance (Ro):


34

For biochemical processes it is usually convenient to consider distances

than transfer rates. For this reason equation 1.10 is written in terms of the Förster

distance R0 at which half the donor molecules decay by energy transfer from

equations (1.9) and (1.10) with ( ) 1−= DT rk τ ,following equation is obtained [78].

( ) ( ) ( ) λλλελπ

dFnN

KR AD

D 4

045

26

0 .128

10ln.9000∫∞Φ

= (1.12)

From this 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

experimentally known value which is accomplished by combining the constant

terms in equation (1.12). If the wavelength is expressed in nm then ( )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= − (1.13)

( )JnKR DΦ×=∴ −− 42560 1079.8 (1.14)

If the wavelength is expressed in cm and J is in units of M-1cm3 then,

( )JnKR DΦ×= −− 422560 1079.8 (in cm6) (1.15)

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

equation.

1.8.4.3 Efficiency of energy transfer:

The energy transfer will be efficient only when the transfer rate is much

faster than the decay rate. If reverse is the case then FRET will be inefficient. The

efficiency of energy transfer (E) is the fraction of photons absorbed by the donor

which are transferred to the acceptor [78]. The fraction is given by


35

( )

( )rk

rkE

TD

T

+= −1τ

(1.16)

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

presence of acceptor. By substituting, equation (1.16) can be rearranged as

66

0

60

rR

RE

+= (1.17)

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 R0. The efficiency

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

The transfer efficiency is typically measured using the relative fluorescence

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

0

1F

FE −= (1.18)

1.8.4.4 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

that of water (n =1.33) or small organic molecules (n =1.39). The quantum yield of

the donor DΦ is determined by comparison with standard fluorophors. The

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

the emission spectrum of the donor with the absorption spectrum of the acceptor,

the higher the value of R0. Acceptors with larger extinction coefficients result in

larger R0 values. The orientation factor K2 is dependent upon geometrical

considerations of emission transition dipole of the donor and the absorption

transition dipole of the acceptor. It is generally assumed equal to 2/3, which is the

value for donors and acceptors that randomize by rotational diffusion prior to

energy transfer [78].


36

1.9 Binding Mechanism:

When the interaction occurs between two different molecules, the binding

parameters like binding constant and number of binding sites can be determined

by following equation [87].

[ ]QnKF

FFlogloglog 0 +=

− (1.19)

where F0 and F have same meaning as discussed earlier, K is the binding constant,

n is the number of binding sites for that particular molecular interaction and [Q] is

the concentration of quencher.

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

determined by plotting the graph of F

FF −0log versus [ ]Qlog . The nature of this

plot will be a straight line with intercept on Y-axis. The slope determines the

number of binding sites while intercept gains the binding constant of that

interaction.

1.10 Thermodynamic parameters:

One can determine the change in free energy (∆G), the entropy change (∆S)

and the enthalpy change (∆H) for any particular interaction by using fluorescence

quenching data. By applying equation 1.19, it is possible to determine binding

constants at various temperatures, with fluorescence quenching measurements.

The fluorescence of the system under study can be recorded at various

temperatures by keeping it in thermostat. The various binding constants are related

with different temperatures by van’t Hoff equation,

R

S

RT

HK

∆+∆−=ln (1.20)

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

temperature, ∆H is change in enthalpy and ∆S is change in entropy accompanying

the molecular interaction.


37

Subsequently, free energy change at different temperatures can be obtained

by Gibb, s equation,

∆G = ∆H –T∆S (1.21)

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 between the different molecules essentially include

hydrogen bond, van der Waals’ forces, electrostatic interactions and hydrophobic

interaction [88]. Ross summed up the thermodynamic laws for estimating the type

of the binding force between organic micromolecule and biological

macromolecule. If ∆H and ∆S are both positive or having some higher values the

main force will be hydrophobic force while if both are negative or some lower

values then hydrogen bond and van der Waals’ forces will be key forces of

interaction and reaction mainly enthalpy driven. Also if ∆H ≈ 0 and ∆S have

positive value then there will be electrostatic force between the acting molecules.

The negative value of ∆G will indicate spontaneity of reaction and vice-versa [89-

92].

1.11 Fluorescence spectrometry:

1.11.1 Instrumentation:

The fluorescence and fluorescence excitation spectra of the solutions of

various drug samples as donor and acceptor molecules in different solvents were

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

the photograph i.e. Figure 1.5. It has following specifications as shown in Table

1.3.

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.


38

Table 1.3:

Instrument : PC based spectrofluorophotometer

Make : JASCO, Japan

Model : FP-750

Light source : 150 W xenon lamp with shielded lamphouse

Monochromator : Holographic grating with 1200 lines/mm

Wavelength range : 220 nm to 730 nm

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

Wavelength accuracy : ±3 nm

Wavelength threw speed : 30,000 nm/min

Wavelength scanning

speed

: 60, 250, 1000, 4000 nm/min

Response : Fast, Medium, Slow, Auto

Sensitivity : Signal to noise ratio of Raman band of water is

higher than 300:1

Photometric display : -999 to +999

Sample chamber : Single cell holder (standard)

Detector : Silicon photodiode for Ex. monochromator and

Photomultiplier tube for Em. monochromator


39

1.11.2 Optical system of FP-750 spectrofluorimeter:

The optical system of the instrument is given in Figure 1.6. 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

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

taken out by the exit slit. A part of the monochromatic light is led to the

monitoring silicon photodiode, SP, by the beam of splitter, BS, while the

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

chamber by the plane mirror M2 and ellipsoidal mirror M3 where it is focused on

the centre of the sample cell. The emission from the sample is focused on to the

entrance slit of the emission monochromator (Em) by ellipsoidal mirror M4 and

two plane mirrors M5 and M6. Monochromatic beam is taken out from the light

dispersed by the diffraction grating G2 of the emission monochromator after going

through the exit slit and is led to photometric photomultiplier tube PMT by the

spherical mirror M7.

1.11.3 Detecting and recording system:

The schematic diagram for the FP-750 system is shown in Figure1.7. 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 wavelengths as well as slit drives

were controlled by the microcomputer.


40

Figure 1.5: The experimental setup of the Spectrofluorimeter


41

Figure 1.6: Optical system of FP-750 Spectrofluorimeter


42

1.11.4 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 monochromator 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) The emission monochromator was then set at the λem and 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.


43

Figure 1.7: System diagram


44

1.11.5 Characteristics of an ideal spectrofluorimeter:

To achieve correct analysis by spectrofluorimetric method, the

components of instrument must posses following characteristics-

1.11.5.1: 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 lamps 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 to Xe arc lamps.

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 this reason, the

holographic gratings are preferable.

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

transmission efficiency of monochromator is dependent upon orientation of

polarizer either vertical or horizontal. The polarization characteristics of

monochromators have important consequences in the measurement of


45

fluorescence. Such measurements must be corrected for the varying efficiencies of

each component.

4: The detector must detect radiations of all wavelengths with equal efficiency.

Almost all fluorimeters use photomultiplier tubes (PMT) as detectors. It is best

regarded as a source of current, which is proportional to the light intensity.


46

1.12 References:

[1] T. Eicher, S. Hauptmann, The Chemistry of Heterocycles, second ed. Wiley-

VCH, Weinheim, (2003).

[2] K. M. Ghoneim, R. J. Youssef, Indian Chem. Soc. 53, (1986), 914.

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