Chapter 1. Introduction
1
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-
Chapter 1. Introduction
2
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
Chapter 1. Introduction
3
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
Chapter 1. Introduction
4
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
Chapter 1. Introduction
5
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
Chapter 1. Introduction
6
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.
Chapter 1. Introduction
7
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
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
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-
Chapter 1. Introduction
8
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
Chapter 1. Introduction
9
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
Chapter 1. Introduction
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.
1.2.4 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
Chapter 1. Introduction
11
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].
Chapter 1. Introduction
12
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:
Chapter 1. Introduction
13
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]
Chapter 1. Introduction
14
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.
Chapter 1. Introduction
15
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
Chapter 1. Introduction
16
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
Chapter 1. Introduction
17
[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
Chapter 1. Introduction
18
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:
Chapter 1. Introduction
19
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
Chapter 1. Introduction
20
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.
Chapter 1. Introduction
21
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 .
Chapter 1. Introduction
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:
Chapter 1. Introduction
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.
Chapter 1. Introduction
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.
Chapter 1. Introduction
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].
Chapter 1. Introduction
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
Chapter 1. Introduction
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
Chapter 1. Introduction
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
Chapter 1. Introduction
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.
Chapter 1. Introduction
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-
Chapter 1. Introduction
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.
Chapter 1. Introduction
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:
Chapter 1. Introduction
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):
Chapter 1. Introduction
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
Chapter 1. Introduction
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].
Chapter 1. Introduction
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.
Chapter 1. Introduction
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.
Chapter 1. Introduction
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
Chapter 1. Introduction
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.
Chapter 1. Introduction
40
Figure 1.5: The experimental setup of the Spectrofluorimeter
Chapter 1. Introduction
41
Figure 1.6: Optical system of FP-750 Spectrofluorimeter
Chapter 1. Introduction
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.
Chapter 1. Introduction
43
Figure 1.7: System diagram
Chapter 1. Introduction
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
Chapter 1. Introduction
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.
Chapter 1. Introduction
46
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