Flourimetry

CONTENTS
 INTRODUCTION
 DEFINITION
 THEORY
 FACTORS AFFECTING FLOURESCENCE
 INSTRUMENTATION
 APPLICATIONS IN PHARMACY

Introduction
 Flourimetry is the measurement of fluorescence at a particular wavelength with the help of
spectrofluorimeter.
 Luminescence is the emission of light by a substance. It occurs when an electron returns
to the electronic ground state from an excited state and loses its excess energy as a photon.
 It is of 3 types.
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 the absorption of light and stops as
soon as the incident light is cut off.
 The substances showing this phenomenon are known as flourescent
substances.

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

TheoryofFluorescenceANDPhosphorescence
A molecular electronic state in which all of the electrons are paired
are called singlet state.
In a singlet state molecules are diamagnetic.
Most of the molecules in their ground state are paired.
When such a molecule absorbs uv/visible radiation, one or more of
the paired electron raised to an excited singlet state /excited triplet
state

Ground excited singlet Triplet state
singlet state spins unpaired
states spin paired
no net mag.field net mag.field

LIGHT EMITING AT ONCE SOURCE STARTS & STOPS WHEM SOURCE STOPS

Nature of molecule
Nature of substituent
Effect of concentration
Adsorption, Light
Oxygen,ph
 Photodecomposition
Temp . &viscosity
Quantum yield
Intensity of incident light
Path length

Nature of molecules
All the molecules cannot show the
phenomenon of fluorescence.
Only the molecules absorbs UV/visible
radiation can show this phenomenon.
Greater the absorbency of the molecule the
more intense its fluorescence.

Nature of substituent
Electron donating group enhances
fluorescence – e.g.:NH2,OH etc.
Electron withdrawing groups decrease
or destroy fluorescence.
e.g.:COOH,NO2, N=N etc.
High atomic no: atom introduced into 
electron system decreases fluorescence.

Fluorescence is directly proportional to
concentration.

FI = Q X Ia
i.e, F = QIOact
Q = Constant for a particular substance
IO = Constant for an instrument
a = Molecular extinction coefficient
t = Path length
C = Concentration of the substance
F = KC Where K represents all constants
FI α Concentration.

 Extreme sensitiveness of the method requires very
dilute solution.
Adsorption of the fluorescent substances on the
container wall create serious problems.
Hence strong solutions must be diluted.

Monochromatic light is essential for the excitation of fluorescence because the intensity will
vary with wavelength.
OXYGEN
The presence of oxygen may interfere in 2 ways.
1] by direct oxidation of the fluorescent substances to non fluorescent.
2] by quenching of fluorescence.
LIGHT

Alteration of the ph of the solution will have significant effect on fluorescence.
Fluorescent spectrum is different for ionized and un-ionized species.
TEMPERATURE & VISCOSITY
Increase in temperature/decrease in viscosity will decrease fluorescence.
PH

Increase in intensity of light incident on sample
Increases fluorescence intensity.
The intensity of light depends upon
1)light emitted from the lamp.
2)Excitation monochromaters.
3)Excitation slit width

The effective path length depends on
both the excitation and emission slit
width.
Use of microcuvette does not reduce the
fluorescence.
Use of microcell may reduce
interferences and increases the measured
fluorescence

QUENCHING
Decrease in fluorescence intensity due to specific effects
of constituents of the solution.
Due to concentration, ph, pressure of chemical
substances, temperature, viscosity, etc

Components of spectroflourimeter
SOURCE OF LIGHT
FILTERS AND MONOCHROMATORS
SAMPLE CELLS
DETECTORS

Sourceof light
MERCURY ARC LAMP. High pressure lamps give lines at 366, 405, 436, 546, 577, 691, 734nm.
Low pressure lamps give additional radiation at 254nm.
XENON ARC LAMP. Spectrum is continuous over the range between over 250-600nm,peak intensity
about 470nm.
TUNGSTEN LAMP. If excitation is done in the visible region this lamp is used.
It does not offer UV radiation.
TUNABLE DYE LASERS

FILTERS
Primary filter-absorbs visible light & transmits uv light.
Secondary filter-absorbs uv radiations & transmits visible light.
MONOCHROMATORS
Exitation monochromaters-isolates only the radiation which is absorbed by the molecule.
Emission monochromaters-isolates only the radiation emitted by the molecule.

The majority of fluorescence assays are carried out in solution.
Cylindrical or rectangular cells fabricated of silica or glass used.
Path length is usually 10mm or 1cm.
All the surfaces of the sample holder are polished in fluorimetry.

PHOTOVOLTAIC CELL
PHOTO TUBE
PHOTOMULTIPLIER TUBES – Best and
accurate.

Multiplication of photo electrons by secondary emission of
radiation.
A photo cathode and series of dynodes are used.
Each cathode is maintained at
75-100v higher than the preceding one.
Over all amplification of 106 is obtained.

Tungsten lamp as source of light.
The primary filter absorbs visible radiation and
transmits uv radiation.
Emitted radiation measured at 90o by secondary filter.
Secondary filter absorbs uv radiation and transmits
visible radiation.

Simple in construction
Easy to use.
Economical
disadvantages
It is not possible to use reference solution & sample solution at a time.
Rapid scanning to obtain Exitation & emission spectrum of the compound is not possible.

Similar to single beam instrument.
Two incident beams from light source pass through
primary filters separately and fall on either sample or
reference solution.
The emitted radiation from sample or reference pass
separately through secondary filter.

 Sample & reference solution can be analyzed simultaneously.
Disadvantage
 Rapid scanning is not possible due to use of filters.

Power
supply
Sourc
e
primary filter
secondary filter
Detector
Sample
cell
Slit
Data
processor

1] Determination of inorganic substances
Determination of ruthenium ions in presence of other
platinum metals.
Determination of aluminum (III) in alloys.
Determination of boron in steel by complex formed
with benzoin.
Estimation of cadmium with
2-(2 hydroxyphenyl) benzoxazole in presence of tartarate.

Field determination of uranium salts.
3]fluorescent indicators
Mainly used in acid-base titration.
e.g.
Fluorescein:colourless-green.
Quinine sulphate: blue-violet.
Acridine: green-violet

Reagent Ion Fluorescence
wavelength
Sensitivity
Alizarin
garnet B
Al3+ 500 0.007
Flavanol
8-Hydroxy
quinoline
Sn4+
Li2+
470
580
0.1
0.2
4] Fluorometric reagent
Aromatic structure with two or more donor functional groups

compound reagent excitation
wavelength
fluorescence
hydrocortisone 75%v/v
H2SO4 in
ethanol
460 520
nicotinamide cyanogen
chloride
250 430
5] Organic Analysis
Qualitative and quantitative analysis of organic aromatic compounds present in cigarette
smoke, air pollutants, automobile exhausts etc.
6] Pharmaceutical Analysis

7] Liquid chromatography
Fluorescence is an imp method of
determining compounds as they appear
at the end of chromatogram or capillary
electrophoresis column.
8]Determination of vitamin B1 &B2.

Flourimetry

In this document

Flourimetry

Editor's Notes

Flourimetry

More Related Content

What's hot

Similar to Flourimetry

More from Zainab&Sons

Recently uploaded

In this document

Flourimetry

Editor's Notes