2. When a beam of light is incident on certain substances they emit
visible light or radiation .This phenomenon is called fluorescence.
Substances showing this phenomenon are fluorescent substances.
This phenomenon is Fluorescence instantaneous i.e start emitting
radiation immediately after the absorption and stops when incident
light is cut off.
Fluorescent substances emit maximum radiation with in 10-10 to 10-
8sec of absorption. This is also called delayed fluorescence
3. When light is incident on certain substances ,they emit light continuously
even after the light is cut off . This is called phosphorescence and such
substances are called as phosphorescent substances.
In the fluorescence process, the electron did not change its spin direction
But under the appropriate conditions, a spin-flip can occur
The light emission process must wait until electron undergoes a spin-flip
to revert back to it original state
Phosphorescent substances emit maximum radiation within 10-4 to 20 sec
or longer.
4. Singlet and triplet stage- A molecular electronic state in which
all electronic spins are paired is called singlet state.
Rate of absorption and emission- rate of absorption is very
rapid and fluorescence emission occur at slower rate.
Deactivation process- the favored route is one that minimize
lifetime of excited state. if deactivation by fluorescence is
rapid w.r.t radiation less procedure is rapid such emission
observed or vice-versa depend on favored rate constant.
5.
6. TERMS INCLUDED -
- vibration relaxation- (occurs in ~ 10-12 s ) collision between molecule of excited
species and those of solvent lead to rapid energy transfer with increase in
temperature.
- Stroke shift- shift towards lower frequency from adsorption band due to
vibration relaxation.
- internal conversion- by which a molecular passes to a lower electronic state
without emission of radiation.
- predissocation observed eg. aliphatic compound
- external conversion- interaction and energy transfer between the excited
molecule and the solvent or solute.
- intersystem crossing- it is a process in which there is a crossover between
electronic state of different multiplicity.
- dynamic quenching (collissional quenching)- nonradiative energy transfer from
an excited species to other molecule is called quenching. And contact between
excited species quenching agent is called dynamic quenching.
7. What Factor Control Whether a Molecule
Fluorescent?
A. Quantum Yield
A. Quantum Yield
f = ________kf______________
kf + ki + kec + kic + kpd + kd
kf = rate constant for fluorescence
ki = rate constant for intersystem crossing
kec = rate constant for external conversion
kic = rate constant for internal conversion
kpd = rate constant for pre-dissociation
kd = rate constant for dissociation
8. B. Structure:
1.) Aromatic
2.) Rigid structures exhibit more
3.) Heavy atoms will decrease
fluorescence
4.) Fluorescence will increase when
molecule is adhered to surface
C. Temperature and Solvent Effects
1.) Lower temperature increases
fluorescence
2.) Solvent contains heavy atoms will
decrease fluorescence but increase
phosphorescence
Factors affecting fluorescence
and phosphorescence
9. Factors affecting fluorescence
and phosphorescence
D. Effect of concentration
E. Adsorption
F. Intensity
G. Oxidation
H. PH
I. Photodecomposition
10.
11. Excitation and emission spectra-
excitation is obtained by measuring luminescence
intensity at fixed wavelength
Because first step in fluorescence is absorption of
radiation and excitation spectra is essential.
12. INSTRUMENTATION
SOURCE - Magnitude of output signal
increase with increase in radiant power
Lamps used-
1 Tungsten or Deuterium lamp
2 Mercury Vapor lamp
3 Xenon arc lamp( continuum)
4 Light emitting diode
Laser used-
1 pulsed nitrogen laser
13. INSTRUMENTATION
Filter and Monochromater- Interference or
adsorption filter have been used in
flourometer for wavelength selection.
Transducer- signal is quite low in intensity so
sensitive transducer are required.
eg. Photomultiplier tubes, charge coupled
devices.
Cells and cell compartments- rectangular
cells of glass or silica.
14. Instrument design of flourometer-
double beam optics for compensating fluctuation in radiant
power.
upper sample beam first passes through excitation
wavelength selector.
transmit radiation that excites fluorescence but exclude
others.
fluorescence observed in right angle to other beams
17. SENSTIVITY: This technique can measure
concentration as low as microgram/ml or even ng/ml.
SPECIFICITY: More specific than absorption
technique.
PRECISION: Up to 1% can easily achieved in
fluorimetery.
Advantages
18. Change in pH affects fluorescent intensity.
Dissolved oxygen may affect fluorescent intensity.
Traces of halides, heavy metals, etc can affect fluorescent
intensity.
19. Applications of photoluminescence methods
1 Determination of uranium salt by flourimetry
2 Determination of inorganic species
-by direct method involve formation of fluorescing chelates.
eg. aromatic structure with two aromatic donor function group
that permit chelate formation with metal ion
-decrease in fluorescing emission because of quenching
8-hydroxy quinoline
benzoin
20. 3 Organic analysis- (adenine, antranilic acid, uric acid,
morphine, proteins etc..)
4 Determination of aspirin in blood serum with high
sensitivity by phosphometric at liq. Nitrogen temperature.
- low conc. of procaine, cocaine, chlorpromazine can be
determined.
5 Determination of Vit B1(Thiamine).
- determination of Vit B2(riboflavin)
21. Comparison of fluorimetry and phosphorimetry with
absorption methods.
Sensitivity of luminescence methods is generally 10 to 103 times
greater than sensitivity of absorption method.
Luminescence method have greater specificity and selectivity than
absorption methods.
22. references
• Willarad H. Hobart ,Instrumental methods of
analysis 17 edition ,CBS publishers and
distributers , New Delhi.
• Skoog DA, West DM, Holler FJ and Crouch S
R, Analytical Chemistry: An Introduction, 7th
ed.
