FLUORIMETRY

UNIT-I (b)
 TOPICS

Theory

Concepts of singlet, doublet and triplet electronic states,

Internal and external conversions,

Factors affecting fluorescence,

Quenching,

Instrumentation and applications

 Introduction

It is an analytical method for detecting and measuring fluorescence in

compounds that uses ultraviolet light, stimulating the compounds and causing
them to emit visible light. The energy/light emitted by the substance has a longer
wavelength than absorbed. This process of emitting radiation with a longer
wavelength than absorbed is known as luminescence.
Fluorescence spectroscopy.
Phosphorescence spectroscopy.
Chemiluminescence spectroscopy
 Fluorescence
• When a beam of light is incident on certain substances they emit visible light or
radiations. This is known as fluorescence.
• Fluorescence starts immediately after light absorption and stops when the incident
light is cut off.
• The substances showing this phenomenon are known as fluorescent substances.
• Analytical technique for identifying and characterizing minute amounts of
substance by excitation of the substance with a beam of ultraviolet/Visible light and
detecting and measuring the characteristic wavelength of fluorescent light emitted.
• Occurs when a molecule absorbs light at one wavelength and re-emits light at a
longer wavelength
• An atom or molecule that fluorescences is termed a fluorophore.
• Fluorometry is defined as the measurement of emitted fluorescence light.
• Fluorometry analysis is a sensitive and widely used quantitative method in chemical
and biological sciences.
Phosphorescence

• When light radiation is incident on certain substances they emit light

continuously even after the incident light is cut off.
• This type of delayed fluorescence is called phosphorescence.
• Substances showing phosphorescence are phosphorescent substances.
• Unlike fluorescence, a phosphorescent material does not immediately
reemit the radiation it absorbs.
• A molecule at rest or in the ground state has three energy levels, i.e., rotational,
vibrational, and electronic.
• When electromagnetic radiation falls on the molecule, it brings changes to its energy
levels during the process of absorption.
• With each electronic level of the ground state, there are many vibrational energy
levels (V0, V1, V2..)
• During radiation absorption, electrons are promoted to excited electronic energy in
one of these vibrational levels.
• Most usual and common is a singlet state (i.e. one in which all electrons are paired,
and in each pair, two electrons spin about their axis in opposite directions)
• The average lifetime of a molecule in the excited state is short, usually 10-8 to 10-6
sec. During this period, molecules at each vibrational level of the excited state would
lose energy.
• Molecule may lose energy by emitting photons or radiations and fall to the original
ground state. The energy of emitted light would be the same as that absorbed.
• The other process may be that usually some molecules initially undergo radiation loss
of energy because of collision and fall to lowest vibrational energy of the excited state.
The vibrational energy is lost to the solvent molecule.
❖From lowest vibration level of excited state, molecule usually return to ground
state by photoemission of energy absorbed and so the emitted radiation is of
longer wavelength. This emitted light is called fluorescence.
❖Thus, fluorescence is defined as radiation emitted in the transition of a molecule
from the lowest level of the singlet excited state to the singlet ground state.
❖In certain cases, the electrons cross over from an excited vibrational singlet to an
excited vibrational triplet state. The triplet state is a metastable electronic level
lower than the singlet state.
❖When this occurs, the lifetime is much longer, often from the fraction of a second
to seconds.
❖The energy emitted from the triplet state to the vibrational ground level is called
phosphorescence.
 Theory
• A molecular electronic state in which all of the electrons are paired is called a
singlet state.
• In a singlet state, molecules are diamagnetic.
• A molecular electronic state in which all of the electrons are unpaired or parallel is
called a triplet state.
• In a triplet state, molecules are paramagnetic.
• Most of the molecules in their ground state are in the singlet state.
• When such a molecule absorbs UV or visible radiation, one or more of the paired
electrons are raised to an excited singlet state.
 Singlet state
• When all the electron spins are paired in the molecular electronic state,
the electronic energy levels do not split when the molecule is exposed
to UV radiation.
 Doublet state
• A doublet state occurs when there is an unpaired electron that gives
two possible orientations when exposed to UV radiation and imparts
different energy to the system.
 Triplet state and triplet excited state
• A singlet or a triplet can form when one electron is excited to a higher
energy level. In an excited singlet state, the electron is promoted in the
same spin orientation as in the ground state (paired).
• In a triplet excited state, the promoted electron has the same spin
orientation (parallel) as the other unpaired electron.
• Singlet, doublet and triplet is derived using the equation for multiplicity, 2S+1,
• S is the total spin angular momentum (sum of all the electron spins).

• Individual spins are denoted as spin up (s = +1/2) or spin down (s = -1/2).

• If we were to calculated the S for the excited singlet state, the equation would be

2(+1/2 + -1/2)+1 = 2(0)+1 = 1, therefore making the center orbital is in singlet

state.

• If the spin multiplicity for the excited triplet state was calculated, we obtain

2(+1/2 + +1/2)+1 = 2(1)+1 =3, which gives a triplet state.

✓ The difference between a molecule in the ground and excited state is that the
electrons is diamagnetic in the ground state and paramagnetic in the triplet state.
✓ This singlet to triplet (or reverse) transition involves a change in electronic state.
For this reason, the lifetime of the triplet state is longer the singlet state by
approximately 10 seconds fold difference.
✓ The radiation that induced the transition from ground to excited triplet state has a
low probability of occurring, thus their absorption bands are less intense than
singlet-singlet state absorption.
✓ The excited triplet state can be populated from the excited singlet state of certain
molecules which results in phosphorescence.
✓ These spin multiplicities in ground and excited states can be used to explain
transition in photoluminescence molecules by the Jablonski diagram.
 Process of excitation and emission
After absorption, a number of vibrational levels of the excited state occur.
The molecules in higher vibration levels of the excited state undergo vibrational
relaxation and reach the lowest vibrational levels of the excited state.
External conversion or collisional deactivation-Decrease fluorescence.
• This process lead to a non-radiative relaxation.
• An excited molecule and solvent or other solutes interact and transfer energy
among each other to deactivate an excited electronic state.
• This process is termed as external conversion or collisional quenching.
• This process is characterized by the solvent effect on fluorescence intensity.
• The condition under which the particle collision reduce lead to enhanced
fluorescence.
• External conversion is a process in which excited molecules lose their energy due
to collisions with other molecules or by transfer of their energy to solvent or
other unexcited molecules. Therefore, the external conversion is influenced by
temperature, solvent viscosity, as well as solvent composition.
 Internal and external conversions
Internal conversion It is an intermolecular
process by which a molecule passes to a
lower energy electronic state without
emission of light.
• Overlap of vibrational energy levels in
two electronic energy levels.
• The process is rapid (10-12 seconds) in
comparison to average lifetime of the
lowest excited singlet state and
therefore, competes with fluorescence.
• IC is an intermediated process which
brings down a molecule a lower energy
electronic state without emitting light.
Intersystem crossing In this process spin of an excited electron is
reversed and change in multiplicity results.
• Molecule in the singlet excited state can cross overt to the triplet
excited state
• The average lifetime of this process is 10-9 second.
• Most common when vibrational manifold overlap exists and when the
molecule has a heavy atom substituent (e.g. Br, I).
• If paramagnetic species (e.g., molecular oxygen) are present in
solution, the intersystem crossing enhances, decreasing the
fluorescence.
Factors affecting fluorescence,
Instrumentation
Source of Light
Sample cells
Detectors
Phototube/
photo-
emissive
Cell
Photomultiplier
Tube
Instruments
Double beam Spectrophotometer
Spectrofluorimeter-Double beam
 Fluorescence Quenching
• Quenching of fluorescence is a physicochemical process that
decreases the fluorescent intensity of light-emitting molecules.
• A variety of molecular associations can result in quenching. These
include excited state reactions, molecular rearrangements, energy
transfer, ground state complex formation, and collisional
quenching.
• Quenching occurs due to various factors like pH, concentration,
temperature, viscosity, presence of oxygen, heavy metals, or
specific chemical substances, etc.
Quenchers
• Reduction of fluorescence intensity of the sample in the presence
of substances other than the fluorescent analyte.
• Example of quenching agents.

Quenching agents Typical fluorophores

Thiocyanate Anthracene
Chloride Quinine
Iodide Tryptophan
Disulfide Tyrosine
Nitric oxide Naphthalene
Types of Quenching

There are four different types of quenching

• Static quenching (contact quenching)

• Dynamic quenching (Collisional)

• Chemical quenching

• Concentration quenching (Self)

Static quenching

Static quenching occurs at the ground state of fluorescing molecule.

• A complex formation occurs between the fluorescing molecule at the

ground state (F) and the quencher molecule (Q) through a strong
coupling. Creating a non-fluorescent complex (F:Q) before excitation or
fluorescing molecule (F).
• Such complex may not undergo excitation or may be excited to a little
extend reducing the fluorescence intensity of the molecule.
• The fluorophore can form a stable
complex with quencher molecule. If
this ground state is non-fluorescent
then the fluorophore has been
statistically quenched.
• Amount of quenching decreases as
temperature increases, as equilibrium
driven to unbound state.
• The quenched state is entirely non
emissive.
• Static quenching occurs at the ground
state of fluorescing molecule.
✓ Caffeine and related xanthines and purines reduce intensity of riboflavin by static
mechanism (Complex formation)
✓ Quenching due to oxygen also follows static quenching mechanism.
Dynamic Quenching (Collisional)

• Collisional quenching occurs by the interaction of a quencher molecule (Q)

with an excited molecule of the fluorescing substance (F*) that facilitate non-
radiative energy transfer from F* to Q without or, less fluorescence.
• Collisional quenching also shortens the
lifetime of fluorophore.
• Halides ions such as chlorides and iodides
are well known collisional quenchers.
• For example, quenching of quinine drug by
chloride ion, or quenching of tryptophan by
iodide ion follow collisional quenching
process.
Chemical Quenching
Chemical quenching is due to various factors like changes in pH, the presence
of oxygen, halides, and electron-withdrawing groups, heavy metals, etc.
Change in pH: Aniline at pH (5-13) gives fluorescence when excited at 290
nm. But at pH<5 or at pH > 13 it does not show any fluorescence.
Oxygen: oxygen leads to the oxidation of fluorescent substances to non-
fluorescent substances and, thus, causes quenching.
Halides and electron-withdrawing groups: Halides like chloride ions, and
iodide ions, and electron-withdrawing groups like –NO2, -COOH, -CHO
groups lead to quenching.
Heavy metals: The presence of heavy metals also leads to quenching because
of collision and complex formation.
Concentration Quenching (Self quenching)

• It occurs when the concentration of the fluorescing molecule increases in a

sample solution.
• The fluorescence intensity is reduced in highly concentrated solution (> 50
µg/mL).
 Applications
✓ Spectrofluorimetry is widely used in pharmaceutical analysis, particularly for the
assay of highly potent drugs present in tiny amounts.
✓ Determination of uranium in salts used extensively in the field of nuclear research.
✓ Estimation of traces of boron in steel by means of the complex formed with
benzene.
✓ Estimation of calcium by fluorimetry with a calcium solution.
✓ Determination of Vitamin B (B1 thiamine and B2 riboflavin) in the food samples
like meat, cereals, etc.
✓ Fluorimetry has been used to check for microbial contamination in foods such as
eggs and dairy products. This technique is of use in detecting organisms that
produce fluorescent pigments (e.g., Pseudomonas fluorescens).
✓ Fluorimetry is employed to carry out both qualitative and quantitative analyses for
various aromatic compounds present in cigarette smoke, air-pollutant, concentrates,
and automobile exhaust.
✓ Quantitative analysis of organic, inorganic and biochemical compounds.
✓ Al, Be, Zn ions combine with 8-hydroxy quinoline forming complexes which
are fluorescent active and can be quantitatively estimated.
✓ Many aromatic compounds e.g., steroids and enzymes have been analyzed by
using fluorescence.
✓ There are fluorescent indicators which are used to detect, correctly the change
in colour of coloured solutions in titrimetry. E.g., Eosin shown a green
fluorescence in this part.
Thank You……..!