INTRODUCTION

DEFINITION
THEORY
FACTORS AFFECTING FLOURESCENCE
INSTRUMENTATION
APPLICATIONS IN PHARMACY
CONCLUSION
REFERENCES

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

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.

 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.

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
singlet
states

excited singlet
state
spin paired
no net mag.field

triplet state
spins unpaired

net mag.field

Fluorescence
Phosphorescence
Radiation

less processes

Vibration

relaxation
Internal conversion
External conversion
Intersystem crossing

LIGHT EMITING AT ONCE SOURCE STARTS &

STOPS WHEM SOURCE STOPS

JABLONSKI ENERGY DIAGRAM

FLUORESCENCE AND
CHEMICAL
STRUCTURE

Fluorescence is most commonly

observed in compounds
containing aromatic functional
groups with low energy.

Most

unsubstituted aromatic
hydrocarbons show fluorescence quantum efficiency increases with
the no: of rings and degree of

CONTD
Simple

heterocyclic do not
exhibit fluorescence.
The n - *singlet quickly converts
to the
n - * triplet and prevents
fluorescence.

Fusion

of heterocyclic nucleus to
benzene ring increases
fluorescence.

Substitution

on the benzene ring

shifts wavelength of absorbance
maxima and corresponding
changes in fluorescence peaks
Fluorescence decreases
with
increasing atomic
no: of the
halogen.
Substitution of carboxylic
acid or
carboxylic group
on aromatic ring inhibits
fluorescence.

Fluorescence is favored in
molecules
with structural
rigidity.

organic chelating agents

complexed with
metal ion

 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

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.

i.e,
Q
IO
a
t
C
F

F
=
=
=
=
=
=

FI = Q X Ia
= QIOact
Constant for a particular substance
Constant for an instrument
Molecular extinction coefficient
Path length
Concentration of the substance
KC Where K represents all constants
FI

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.

 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.

fluorescence quantum
yield:

Kf =
kec =
kic =
kisc =
kpd =
Kd =

fluorescence
external conversion
internal conversion
intersystem crossing
pre dissociation
dissociation

 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

 Decrease

in fluorescence intensity
due to specific effects of constituents
of the solution.
Due to concentration, ph, pressure of
chemical substances, temperature,
viscosity, etc.
Types of quenching
Self quenching
Chemical quenching
Static quenching
Collision quenching

Fluorescence

Fluorescence

Calibration curve
(Low con)

calibration curve
(High con)

Concentration of

Concentration of

fluorescing species

fluorescing species

Deviations at higher concentrations can be

attributed to self-quenching or selfabsorption.

 Here

decrease in fluorescence intensity due

to the factors like change in ph,presence of
oxygen, halides &heavy metals.

ph- aniline at ph 5-13 gives fluorescence

but at ph <5 &>13 it does not exhibit
fluorescence.

halides like chloride,bromide,iodide &

electron withdrawing groups like no2,cooH
etc. leads to quenching.

Heavy metals leads to quenching,

because of collisions of triplet ground state.

 This

occurs due to complex formation.

e.g.. caffeine reduces the fluorescence of
riboflavin by complex formation.
COLLISIONAL QUENCHING

It

reduces fluorescence by collision. where

no. of collisions increased hence quenching
takes place.

Fluorescence mainly classified in to 2

categories.

Based on wavelength of emitted radiation

Stokes fluorescence
Antistokes fluorescence
Resonance fluorescence

Based on phenomenon
Sensitized fluorescence
Direct line fluorescence
Stepwise fluorescence
Thermally assisted fluorescence.

Based upon wavelength:

stokes fluorescence: wavelength
of emitted radiation is longer
than absorbed radiation.
Anti stokes: wavelength of
emitted radiation is shorter than
absorbed radiation.
Resonance fluorescence:
wavelength of emitted radiation
is equal to absorbed radiation.

 Sensitized

fluorescence-

When elements like

thalium,zn,cadmium or an alkali metal
are added to mercury vapour these
elements are sensitized and thus gives
fluorescence.
Direct

line fluorescence

Even after the emission of

radiation, the molecules retain in
metastable state and finally comes to
the ground state after loss of energy by
vibrational transmit.

 Stepwise

fluorescence
this is conventional type of fluorescence
where a part of energy is lost by vibrational
transition before the emission of fluorescent
radiation.
Thermally assisted fluorescence
here excitation is partly by
electromagnetic radiation and partly by
thermal energy.

INSTRUMENTATION

 SOURCE

OF LIGHT

FILTERS

AND MONOCHROMATORS

SAMPLE

CELLS

DETECTORS

 MERCURY

ARC LAMP.
XENON ARC LAMP.
TUNGSTEN LAMP.
TUNABLE DYE LASERS.

MERCURY ARC LAMP

Produce intense line spectrum above

350nm.
High pressure lamps give lines at
366,405, 436, 546,577,691,734nm.
Low pressure lamps give additional
radiation at 254nm.

Intense radiation by passage of current

through an atmosphere of xenon.
Spectrum is continuous over the range
between over 250-600nm,peak intensity
about 470nm.

Intensity

of the lamp is low.

If

excitation is done in the

visible region this lamp is
used.

It

does not offer UV radiation.

Pulsed

nitrogen laser as
the primary source.

Radiation

in the range
between 360 and 650 nm
is produced.

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
PHOTO

CELL

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 10 6 is
obtained.

SINGLE

BEAM FLUORIMETER

DOUBLE

BEAM FLUORIMETER

SPECTROFLUORIMETER(DOUBLE

BEAM)

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.

Sample cell
Power Source primary filter
supply

Slit
secondary filter

Detector

Data processor

The primary filter in double beam

fluorimeter is replaced by excitation
monochromaters.

The secondary filter is replaced by

emission monochromaters.

The incident beam is split into sample

and reference beam using a beam
splitter.

The detector is photomultiplier tube.

Advantages
Rapid

scanning to get Exitation &

emission spectrum.
More sensitive and accuracy when
compared to filter fluorimeter.

Power Source Excitation

monochromator
supply

Sample
cell
Emission
monochromator
Detector
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.:
eosin- colorless-green.
Fluorescein:colourless-green.
Quinine sulphate: blue-violet.
Acridine: green-violet

4] Fluorometric reagent
Aromatic structure with two or more
donor functional groups

Reagent

Ion

Fluorescen Sensitivi
ce
ty
wavelengt
h

Alizarin
garnet B

Al3+

500

0.007

Flavanol

Sn4+

470

0.1

8-

Li2+

580

0.2

5] organic analysis
Qualitative and quantitative analysis of
organic aromatic compounds present in
cigarette smoke, air pollutants,
automobile exhausts etc.

6] pharmaceutical analysis

compound

reagent

excitation
wavelengt
h

fluorescence

hydrocortiso
ne

75%v/v
H2SO4 in
ethanol

460

520

nicotinamide

cyanoge
n
chloride

250

430

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.

Fluorimetry ,nowadays can be

used in detection of impurities in
nanogram level better than
absorbance spectrophotometer
with special emphasis in
determining components of
sample at the end of
chromatographic or capillary
column.

 Douglas

A Skoog, Principles of
instrumental analysis

H:\UV-Vis

Luminescence Spectroscopy Theory.mht

Dr.B.K.Sharma,

Instrumental
methods of chemical analysis
Gurdeep R Chatwal, Instrumental
methods of chemical analysis

http://en.wikipedia.org/wiki/Fluorescence

http://images.google.co.in/imghp?
oe=UTF-8&hl=en&tab=wi&q=fluorescence

http://www.bertholdtech.com/ww/en
pub/bioanalytik/biomethods/fluor.cfm