SPECTROFLUORIMETRY (Theory Principle Factors Affecting Fluorescence)

Still, the use of spectro-fluorimetry as an analytical tool provides a well-defined identity of the compounds present in the sample based on their unique fluorescent nature.

1. (Spectrofluorimetry /
Fluorescence
Spectroscopy)



Prepared by
Prof. (Dr.) Dinesh Kumar Mehta
MMCP, (MMDU), Mullana

2. Introduction
Although some information about molecular structure may be
Derived from excitation and emission spectra, qualitative application
of spectrofluorimetry are rare and vast majority of applications
in pharmaceutical analysis.
Still the use of spectrofluorimetry as an analytical tool provides a well-
defined identity of the compounds present in the sample on the basis
of their unique fluorescent nature.
 Luminescence is emission of light by a substance not resulting from
heat is thus a form of cold body radiation. It can be caused by chemical
reactions, electrical energy, subatomic motions, or stress on a crystal.
Types of luminescence
a) By Mechanism:
i) Fluorescence Spectroscopy
ii) Phosphorescence Spectroscopy
b) By Excitation Source:
i) Chemiluminescence
ii) Cathodoluminescence

3. iii) Electroluminescence
iv) Photoluminescence
Fluorescence
 Fluorescence is the phenomenon where by a molecule, after
absorption radiation, emits radiation of a longer wavelength
 A compound absorbs radiation in the UV – region and emits visible
light. Absorption of UV /Visible radiation causes transition of
electrons from ground state (low energy) to excited state (high
energy).
Fluorescence examples
There are some materials that emit light on irradiation with light. When
light is incident on fluorite (CaF2), it emits blue light. Similarly,
chlorophyll emits red light when dissolved in ether. Some examples of
fluorescent substances are given as:
 Organic dyes like Fluorescein, and Eosin.
 Quinine sulfate emits blue light.
 Some glasses emit different colors when an x-ray falls on them.

4. Principle
 Fluorescence is an emission phenomenon where an energy transition from
a higher to a lower state is accompanied by radiation, after absorption
radiation, emits radiation of a longer wavelength
 A compound absorbs radiation in the UV – region and emits visible light.
Absorption of UV/visible radiation causes the transition of electrons from
the ground state (low energy) to the excited state (high energy).
 Only molecules in their excited forms are able to emit fluorescence; thus
they are to be brought into a state of higher energy prior to the emission
phenomenon.
Important Terms
Singlet ground state: state in which electrons in a molecule are paired.
Singlet excited state: state in which electrons are unpaired but of opposite
spins.
Triple state: state in which unpaired electrons of some spin are present.
Excitation process: absorption of energy or light followed by a conversion
from the ground to an excited state.
Relaxation process: the process by which atom or molecule loses energy
& and returns to ground state.

6. Difference Between Fluorescence and Phosphorescence
1. The main difference between fluorescent and phosphorescent
is, that fluorescence is the absorption of energy by atoms or
molecules followed by immediate emission of light or
electromagnetic radiation. On the other hand,
phosphorescence is the absorption of energy by atoms or
molecules followed by delayed emission of electromagnetic
radiation.
2. Fluorescent emission of radiation or light suddenly stops on
the removal of the source of excitation. On the other hand, the
phosphorescence emission of radiation remains for some time
even after the removal of the source of excitation.
3. If we distinguish between fluorescence and phosphorescence,
the time period or interval between absorption and emission is
the primary factor. It is very short for fluorescence and
comparatively long for phosphorescence.
4. Another differentiate between fluorescence and
phosphorescence is the emitted photon (light) has lower
energy than the absorbed photon and emission occurs at a
longer wavelength than the incident light in fluorescence and
the longer wavelength than fluorescence in phosphorescence.
5. In fluorescent materials, gives an “an immediate flash or
afterglow” on excitation. And the phosphorescent materials

7. appear to “glow in the dark” because of the slow emission of
light over time.
6. Fluorescence and phosphorescence difference is the excited
atom has a comparatively short lifetime before its transition to
a low energy state in fluorescence, and for phosphorescence,
the excited atom has a comparatively long time before its
transition to a low energy state.

Factors affecting fluorescence
Conjugation: Molecule must have unsaturation i.e. it must have π
electrons so that UV/vis radiation can be absorbed. If there is no
absorption of radiation, there will not be fluorescence.
Rigidity of structures
Rigid structures will produce more fluorescence, while flexible structure
will produce less fluorescence.
Nature of substituent groups
Electron donating groups like amino (NH2), hydroxyl (OH) groups
enhance fluorescence activity. Electron withdrawing groups like Nitro
(NO2), carboxylic group (COOH) reduce fluorescence. Groups like SO3H
or on NH4
+
have no effect on fluorescence intensity.

8. Effect of temperature
Increase in temperature leads to increase in collisions of molecules and
decrease in fluorescence intensity while decrease in temperature leads to
decrease in collisions of molecules and increased fluorescence intensity.
Viscosity
Increase in viscosity leads to decreased collisions of molecules which will
enhance fluorescence intensity while decrease in viscosity causes
increased collisions of molecules which cause decreased fluorescence
intensity.
Oxygen
Oxygen decreases the Fluorescence intensity in two ways: Oxidises
fluorescence substances to non-fluorescence substances. It quenches
fluorescence because of paramagnetic properties.
Effect of pH
a. Aniline: Neutral or alkaline medium shows visible fluorescence while
acidic conditions give fluorescence in UV region only.
b. Phenols: Acidic conditions do not give fluorescence while alkaline
conditions gives good fluorescence

9. Fluorescence quenching
The intensity of fluorescence radiation may be reduced or prevented when
a photo chemically excited atom has a chance to collide with another atom
or molecule before it fluoresces. This process is called quenching of
fluorescence.
When an atom or molecule is photo chemically excited, this excited atom
has a chance to collide with another ground state atom of other chemical
species. In such collision, energy is transferred to the atom of other species
due to collision, and therefore, the excited atom returns to its ground state
without emitting light radiation. This process is termed as Fluorescence
quenching.
If only a fraction of the energy of the excited atom of fluorescent material
is transferred to other non-fluorescent species, then the energy of
fluorescent species is decreased. In such a case, fluorescence with reduced
intensity is observed.