ppt of spectrofluorimety topic of instrumental method of analysis.

1. SPECTROFLUORIMETRY
BY-RANA RUDH PRATAP SINGH

2. CONTENT
 INTRODUCTION
 THEORY
 INSTRUMENTATION OF FLUORESCENCE
 FACTORS AFFECTING FLUORESCENCE
 QUENCHING
 TYPES OF QUENCHING
 APPLICATION

3. INTRODUCTION
 Absorption of uv-visible radiation causes transition
of electrons from ground state to excited state.
 As excited state is not stable so,excess energy is
released and the electron shifts from excited state to
ground state.
 This measurement or study of emitted radiation is the
principle of flourimetry.
 Phosphorescence is the delay of fluorescence,which
means the emittion of radiation when electron
undergoes transition from triplet state to ground
state.

4. FLUORESCENCE AND FLOURIMETRY
FLUORESCENCE
 When a beam of light is incident on certain
substances,they emit visible radiation .This is known as
fluorescence.
 Fluorescence starts immediately after the absorption of
light and stops as soon as the incident light is cut off.
 The substance showing this phenomenon are known as
fluorescent substances.
FLUORIMETRY
 Fluorimetry is an analytical method for the measurement
of fluorescence, based upon the emission of absorbed
radiation by the molecules.
 The instrument used is called as spectrofluorimetry.

6. THEORY OF
FLUORESCENCE
 A molecule at ground state is said to posses
three different types of energy levels.
a) Rotational energy
b) Vibrational energy
c) Electronic energy
 When an electromagnetic radiation falls on
the molecule it eventually brings the
changes in the energy levels during the
process of absorption.

7.  The main theory involved in the flourescence is
when an incident light absorbed by the sample ,it
undergoes the transition from the singlet ground state
to singlet excited state.
 Where the singlet exited state is not stable and the
molecules present in exited state immediately return
to the ground state by emitting the energy.
 A part of energy is lost due to vibrational transition
and the remaining energy is emitted as uv/visible
radiation o longer wavelength than the incident light.
Absorbed wavelength radiation excitation
wavelength
Emitted wavelength radiation emission
wavelength

8. INSTRUMENTATION OF FLUORESCENCE
 SPECTROPHOTOMETER
1) Source of light
2) Monochromator
3) Sample cells
4) Detectors
5) Recorder

9. FACTORS AFFECTING FLUORESCENCE
1) Effect of substitution
2) Nature of molecule
3) Effect of concentration
4) Temperature
5) Viscosity
6) Oxygen
7) Path length
8) Intensity of incident light

10. QUENCHING
 Quenching refers to any process that reduces
the fluorescence intensity of a given sample.
 It occurs due to various factors like
concentration,temperature,presence of
oxygen,viscosity,etc.
TYPES OF QUENCHING
a) Self quenching or concentration quenching
b) Chemical quenching
c) Collisional quenching

11. APPLICATION
1. Determination of inorganic substances like
aluninium ion,zinc,tin ion.
2. Determination of organic substances like
aromatic polycyclic
hydrocarbon,naphthols,steroids,protein.
3. Pharmaceutical applications
 separate the mixture of medicinal substance

12. THANK YOU