Fluorimetry

• Introduction
• Theoretical principle
• Concepts of singlet, doublet and triplet electronic states,
• internal conversions, and intersystem crossion
• Factors affecting fluorescence,
• Quenching
• Instrumentation
• Applications
 Introduction
• Fluorescence : It is a phenomenon of emission of radiation when the
molecules are exited by radiation at certain wavelength. Emission takes
place at longer wavelengths than that of the excitation radiation
• excitation is brought about by absorption of photons. As a consequence,
the phenomenon is referred as photoluminescence.
• Fluorimetry:- It is measurement of fluorescence intensity at a particular
wavelength wit the help of a filter fluorimeter or a spectrofluorimeter.
• Fluorescence occurs in simple as well as in complex gaseous, liquid, and
solid chemical systems.
• This techniques is very sensitive (Very dilute solutions can be analyzed)
• Another advantage of photoluminescence methods is their large linear
concentration ranges, than that of absorption methods.
 Some Basic terminologies involved in fluorescence
• Molecule contains σ electrons, π electrons and non bonding (n) electrons.
• The electrons may be present in bonding molecular orbital. It is called as highest occupied
molecular orbital (HOMO).It has least energy and are more stable.
• When the molecules absorbs radiant energy from a light source, the bonding electrons may be
promoted to anti bonding molecular orbital, i.e. lowest un- occupied molecular orbital(LUMO). It
has more energy and hence less stable.
• The process of promotion of electrons from HOMO to LUMO with absorption of energy is called
as excitation.
a. Singlet state:-a state in which all electrons in a molecule are paired ↑↓
b. Singlet excited state:- a state in which electrons
are paired and of opposite spin like ↑↓
c. Triplet state:- a state in which unpaired
electrons of same spin present ↑ ↑
d. Doublet state:- a state in which unpaired electrons
is present either as ↑or ↓
• Singlet state At the ground state , the molecular orbitals are occupied by two
electrons. the spins of the two electrons in the same orbital must be antiparallel.
This implies that the total spin, S, of the molecule in the ground state is zero [½ +
( ½)].
• This energy state is called “singlet state” and is labeled as S0
• After absorption of radiation, the electron spins go to the the excited state-
1. where the spin is antiparallel. This state is a singlet (antiparallel) state.
2. where the spin is parallel. This state is a triplet (parallel) state.
• The absorption of a photon of suitable energy causes the molecule to get excited
from the ground state to one of the excited states. This process is called as excitation
or activation and is governed by Franck‐Condon principle.
• excitation/activation is followed by deactivation which is categorized by-
• Nonradiative deactivation – not accompanied by any radiative loss
• Radiative deactivation- emission of radiation
• There are three different nonradiative means of deactivation.
1. vibrational relaxation, In the higher vibrational levels of an excited state, the
molecule rapidly loses (in < 10-12 s) its excess vibrational energy by collision
with other molecules and falls to the lowest vibrational level of the excited
state. In this nonradiative mode of relaxation, called vibrational relaxation,
the energy lost is dissipated as heat to the surroundings.
2. Internal Conversion Once the molecules that are excited to an electronic
state (say S2) higher than the S1 state, reach the vibrational ground state in
the electronic level, these can pass to a higher vibrational level of a lower
excited state (S1) which has the same energy.
3. Intersystem Crossing In rare occasions, the molecule in the vibrational states
of a singlet excited state may cross over to a vibrational level of a triplet
state if the two have same energy. This process is called intersystem
crossing. This spin-exchange mechanism may also lead, though very rarely, to
the crossing over from a triplet state to a singlet state.
Radiative Deactivation :
1. Fluorescence: When the molecule in the excited state (S1) relaxes down
to the lowest vibrational level it may emit a photon and come down to
the electronic ground state (S0). This process is called fluorescence and
takes about 10‒9 s.
Another radiative relaxation process can arise from the excited molecule
that had crossed over to the triplet excited state by intersystem crossing and
has relaxed to the vibrational ground state in the triplet excited state.
2. Phosphorescene: From the lowet vibrational level of triplet excited
state, the molecule emits a photon and comes down to a vibrational
mode of the electronic ground state, S0. This phenomenon is called
phosphorescence.
Fluorescence_ Theoretical Principle

• When light of appropriate wavelength is absorbed by a molecule the electrons

are promoted from singlet ground state to singlet excited state. once the
molecule is in this excited state relaxation can occur via several process. The
process are as follows –
1) Collisional deactivation
2)Fluorescence
3)Phosphorescence.
Fluorescence phenomenon can be explained by Energy-Level Diagram,called a
Jablonski diagram.
The lowest heavy horizontal line represents the ground-state energy of the
molecule which is normally a singlet state, and is labeled So. At room
temperature, this state represents the energies of most of the molecules in a
solution.
Jablonski diagram  The upper heavy lines are
energy levels for the ground
vibrational states of three
excited electronic states.
 The two lines on the left
represent the first (S1) and
second (S2) electronic singlet
states.
 The one on the right
(T1)represents the energy of
the first electronic triplet state.
The energy of the first
excited triplet state is lower
than the energy of the
corresponding singlet state.
• Numerous vibrational energy levels are associated with each of the four electronic
states. as suggested by the lighter horizontal lines. As shown in Figure , absorption
transitions can occur from the ground singlet electronic state (S 0) to various
vibrational levels of the excited singlet electronic states (S1 and S2).
• a transition of energy from the lowest vibrational level of an excited singlet
electronic state to vibrational states of ground singlet electronic state S0 is called
fluorescence and
• a transition from the lowest vibrational level of triplet state to vibrational states of
ground singlet electronic state S0 is called Phospharescence.
• Note that direct excitation to the triplet state is not shown. Because this transition
involves a change in multiplicity. it has a very low probability of occurrence. A low
probability transition of this type is called a forbidden transition.
• Molecules excited to electronic states S1 and S2, rapidly lose any excess vibrational
energy and relax to the ground vibrational level of that electronic state. This non
radiational process is termed vibrational relaxation.
 FACTORS AFFECTING FLUORESCENCE
The common factors affecting the fluorescence are as follows. –
1. Concentration of substance 2. Temperature 3. pH 4.Dissolved oxygen
5. Solvent 6. Solvent viscosity 7. Solvents with heavy atoms

1. Concentration of substance: fluorescence is best observed in dilute solutions. In

concentrated solutions, fluorescence intensity is reduced and is not quantitative. This is
called concentration quenching. The reason is that in dilute solutions, absorbed light is
distributed equally through the entire path length of the solutions. But in concentrated
solutions, first part of the solution in the path absorbs more and then less radiations
are available for the rest path. In concentrated solutions, the fluorescence decreases
because of –
• Collisions between molecules causes vibrational relaxation and loss of
fluorescence
• Emitted fluorescence may be reabsorbed in concentrated solutions.
2. Temperature FACTORS AFFECTING FLUORESCENCE
• A rise in temperature is almost always accompanied by a decrease in fluorescence.
• The change in temperature causes the viscosity of the medium to change which in turn changes
the number of collisions of the molecules of the fluorophore with solvent molecules.
• The increase in the number of collisions between molecules in turn increases the probability for
deactivation by internal conversion and vibrational relaxation.

3. pH : Relatively small changes in pH can sometimes cause substantial changes in the fluorescence
intensity and spectral characteristics of fluorescence.
e.g., serotonin shows a shift in fluorescence emission maximum from 330 nm at neutral pH to 550
nm in strong acid without any change in the absorption spectrum.
• In the molecules containing acidic or basic functional groups, the changes in pH of the medium
change the degree of ionisation of the functional groups. This in turn may affect the extent of
conjugation or the aromaticity of the molecule which affects its fluorescence.
• e.g. , aniline shows fluorescence while in acid solution it does not show fluorescence due to the
formation of anilinium ion.
• Therefore, pH control is essential while working with such molecules and suitable buffers should
be employed for the purpose.
 FACTORS AFFECTING FLUORESCENCE

4. Dissolved oxygen
• The paramagnetic substances like dissolved oxygen and many transition
metals with unpaired electrons dramatically decrease fluorescence and cause
interference in fluorimetric determinations.
• The paramagnetic nature of molecular oxygen promotes intersystem crossing
from singlet to triplet states in other molecules. • The longer lifetimes of the
triplet states increases the opportunity for radiationless deactivation to occur.
• Presence of dissolved oxygen influences phosphorescence too and causes a
large decrease in the phosphorescence intensity. • It is due to the fact that
oxygen which is in triplet state at the ground state gets the energy from an
electron in the triplet state and gets excited. • This is actually the oxygen
emission and not the phosphorescence.
• Therefore, it is advisable to make phosphorescence measurement in the
absence of dissolved oxygen.
 FACTORS AFFECTING FLUORESCENCE
5. Solvent
The changes in the “polarity” or hydrogen bonding ability of the solvent may
also significantly affect the fluorescent behaviour of the analyte.
The difference in the effect of solvent on the fluorescence is attributed to the
difference in their ability to stabilise the ground and excited states of the
fluorescent molecule.
6. Solvent viscosity
Increased viscosity increases fluorescence as the deactivation due to
collisions is lowered.
7. Solvents with heavy atoms :
Presence of heavy atom decreases fluorescence
phosphorescence increases due to the presence of heavy atoms in the
solvent.
 Photoluminescence and Structure
• The presence of the benzene ring and the nature of substituents on it seem to favor
the fluorescent behavior of the molecule.
• The halogen substituents tend to decrease the fluorescence and shift the
fluorescence bands to longer wavelengths; the effects increase with increase in the
atomic mass of the substituted halogen.
• Compounds with fused ring are found to be especially fluorescent,
• The extent of fluorescence is found to be directly proportional to the number of
rings in the molecule
• The structural rigidity in a molecule favors fluorescence.
• Aliphatic and alicyclic carbonyl compounds or highly conjugated double bond
structures also show fluorescence.
• The fluorescence observed with
rigid cyclic molecules with pi‐
bonds is found to be enhanced by
electron donating groups e.g.,
−NH2, OR, –OH and OCH3,
• The electron withdrawing groups
such as COOH, NO2, N=N and Br,
I and CH2COOH tend to reduce it.
• On the other hand the nonrigid
molecules do not fluoresce much,
as these rapidly lose the absorbed
energy through nonradiative means
like, vibrational relaxation or even
degradation.
• Another observation is that the simple heterocyclic compounds like
pyridine, furan, thiophene and pyrrole do not fluoresce significantly. but
when fused to a benzene ring, it becomes highly fluorescent.
• For example, the heterocyclic pyridine is nonfluorescent, but quinoline
and isoquinoline in which the pyridine ring is fused to a benzene ring are
highly fluorescent.
 FLUORESCENCE QUENCHING
The fluorescence emission is quite sensitive to the presence of impurities and other
species in the sample. These cause a decrease in the intensity of fluorescence
emission.
Quencinging: The decrease in the fluorescence intensity arising out of the
interaction of the excited state of the fluorophore with its surroundings is called
quenching.
e.g. quinine fluorescence is quenched by the presence of halide ions.
Mechanisms of quenching:
1. collisions between excited and ground state molecules leading to an increase in
the amount of radiationless relaxation. It is called self-quenching and it alters
the ratio of excited molecules that relax via the fluorescence pathway. Since
self-quenching depends on the rate at which collisions occur, it increases with
an increase in the concentration of the analyte. Due to selfquenching, the
photoluminescence efficiency varies with the concentration.
2. Another mechanism that leads to the decrease in fluorescence intensity is called
self-absorption or the inner-cell effect. It is observed in the molecules in which
the absorption band overlaps with the wavelength of the emitted (fluoresced)
photon. In such a situation some of the emitted photons are reabsorbed before they
can escape the solution. This is called self-absorption. this is also significant at
high analyte concentrations.
3. The intermolecular electronic energy transfer from the excited molecule to a
quencher molecule is one of the common ways by which the fluorescence quenching
occurs. The process can be represented as follows.
M* + Q Q* + M
where, the excited analyte molecule (M*) transfers its excitation energy to a quencher
molecule Q, whereby it gets de-excited to M forming an excited quencher molecule,
Q*.
In case Q* happens to be a fluorescent species, then its fluorescence is called
sensitised fluorescence.
INSTRUMENTATION_FLUORIMETRY
• In fluorescence instruments, the same
components are used in absorption
spectrophotometers. However, the
geometric arrangement of the
components is somewhat different. This
is due to the reason that any
transmitted radiation is not measured
along with the fluorescence.
• The absorption or transmission of
radiant energy occur only along the
direction of the incident light.
• whereas the fluorescence radiation emanates in all directions. The detection
of transmitted radiation is avoided by placing the detector at a right angle to
the transmitted beam, as shown in Fig
 Schematic diagram of Fluorimeter
The essential components of
an instrument used to
measure fluorescence of the
sample are:
1. Excitation light sources
2. Filters or
Monochromators
3. Sample holder
4. Detector
5. Readout device/output
device
1. Excitation light Sources
• the fluorescence power or intensity, IF, is directly proportional to the source
power, P0. An increase in P0 will produce a larger signal for a given
concentration and thereby improve sensitivity. Therefore, the source must be
more intense than that required for UV-VIS absorption spectroscopy.
• in fluorescence measurements a source with intense emission lines at certain
frequencies are desirable.
• For Filter fluorimeters often employ a low-pressure mercury vapor lamp. It
produces intense lines at certain wavelengths.
a. mercury vapor is encased in a glass or used silica envelope at low pressure
and excitation of mercury atoms is done by electric discharge. Principle lines of
wavelength are emitted. By isolating one of the principle line at 366, 405, 436,
520, or 580 nm high intensity radiation can be isolated using primary filter.
• DEMERIT: Not suitable for continuous spectral studies,(because it doesn’t give
continuous radiations).
b. xenon arc lamp – used for Spectrofluorometers It possesses two tungsten
electrodes separated by some distance 8mm). These are enclosed in a glass tube
(for visible) with quartz or fused silica. The xenon gas is filled under pressure
(10-30atm). An intense arc is formed between electrodes by applying high
voltage,
Advantage: a continuous radiation source,. These produce an intense
continuum between about 250 and 600 nm.
Disadvantage: lamp produces lot of heat, the lamp assembly needs to be cooled,
therefore, these instruments cannot be used for routine work.
c. laser excitation source. A tunable dye laser, using a pulsed nitrogen laser as
the primary source can produce monochromatic radiation between 360 and 650
nm.
2. Wavelength Selectors: it consist of lenses made of glass or quartz, used to focus
radiation, entrance slits and filters/monochromators.
Depending upon type of instrument either filters(for fluorimeters) or
monochromators (for spectrometers)are used.
For fluorimenters, filters are used-
1. Excitation/Primary filter: placed after radiation source and before sample. It
isolates excitation radiation form radiation source, which excites sample
molecules.
2. Emission/Secondary filter: it is placed after sample, at right angle to the incident
radiation. It isolates emission/fluorescences radiation.
Filters are of two types- a. absorption filters & b. interference filters
• Absorption filters are comprised of a suitably absorbing substance dispersed in
gelatin, glass or plastic.
• interference filters having high transmission are used for sophisticated instruments
• For spectrofulorometers diffraction gratings are used.
3. Sampling: fluorescence measurements of the analyte are carried out in
solution. For this the sample is taken in a cuvette or in a flow cell.
The cuvettes generally are circular, or square shaped. These are constructed of a
material that will pass both the incident as well as the emitted light through it.
Glass (visible radiations) and quartz (UV Radiatons) cuvettes are generally used.
The square and circular cuvettes can be used. Square cuvettes has 10mm path
length. Cuvettes have leads to cover to avoid loss of volatile samples.
The sample in the cuvettes absorbs excitation radiation and then emits
fluorescence in all direction. It is allowed to pass through the secondary filter,
placed at right angles to collect emitted radiation and avoid interference of
transmitted radiation.
After passing through secondary filter, radiations are collected by detector.
4. Detector: the basic requirement for the detector is that it should be able to detect
weak optical signals. It has following reasons.
• The fluorescence signals for an analyte present at low concentration is weak. It is
due to low photoluminescence efficiencies and because only a small fraction of the
fluorescence radiation reaches the detector.
Photomultiplier Tube Detector: Principle of operation-multiplication of photoelectrons
by secondary emission of electrons
In a vacuum tube, a primary photo-cathode is fixed which
receives radiation from the sample.
Some eight to ten dynodes are fixed each with increasing
potential of 75-100V higher than preceding one.
Near the last dynode is fixed an anode or electron
collector electrode.
Photo-multiplier is extremely sensitive to light and is best
suited where weaker or low radiation is received
• 5. Signal processor/Read out Devices
• The output from the detector is suitably amplified and displayed on a read
out device like a meter or digital display. The sensitivity of the amplifier
can be changed so as to be able to analyse samples of varying
concentrations. In some instruments the display can be adjusted to directly
give the output in terms of the concentration
 Applications of fluorimetry

• It is widely used in the qualitative and quantitative analysis of inorganic

compounds and has become extensively used in spectroscopic technique in
the fields of Biochemistry and Molecular Biophysics also.
• Fluorimetry is used in the research and developments for analysis of vitamins
(vitamin A), drugs (Amphetamine, Codeine, Morphine).
• Fluorimetry is used for diagnostic purposes (scanning of some internal body
parts using some fluorescent dyes )
• It is used in the environmental study (Polyaromatic hydrocarbons)
• Used for the analysis of water and soil samples.
• If some drugs are not able to detect, the fluorescent derivatives of the same
can be prepared for analyzing same.