1. Fluorescence

2. Topics
Definition
Theory’s
Factor’s
Instrumentation
Applications

3. Luminescence
Emission of previously absorbed radiation

4. Types :
Luminescence is two types
1. Florescence
2.Phosphorescence

5. Fluorescence
When a beam of light is incident on certain substances, they emmit visible light or radiations.
( The substance is fluorescent substance. Measurement is called as fluorimetry. )

7. Phosphorescence
When a beam of light is incident on certain substances, they emit radiations continuously even after the incident light is cut off .
(The substance is phosphorescent. Measurement of radiations is Phosphorimetry. )

9. Types of fluorescence
1. Based upon the wave length of emitted radiation when compared to absorbed radiation
- Stokes's fluorescence
- Anti- Stokes's fluorescence
- Resonance fluorescence

10. 2. Based upon the phenomenon
- Sensitized fluorescence
- Direct line fluorescence
- Stepwise fluorescence
- Thermally assisted fluorescence

11. Theory 1.Singlet and triplet states
2.Excited-state state process in molecules
3.Relation between fluorescence intensity and concentration
4.Factors affecting fluorescence

12. Singlet and triplet states
A molecular electronic state in which all of the electron spins are paired is called a singlet state. (spine state)
If ‘no’ un pair electrons are present (n=0), there is only n+1 or 0+1 spin state. Such state is called a singlet state. Similarly, systems having 1,2,3,4…… unpaired electrons refer to doublet, triplet, quartet etc. state respectively

13. Excited –state processes in molecules
1.Collisional deactivation
2.Fluorescence
3.phosphorescence

14. processes in molecules
Absorption
Collisional deactivation
Fluorescence
Phosphorescence
Intersystem crossing
Vibrational relaxation v0 v3 v5 v7 v0 v3 v5 v7 S1 T v0 v3 v5 v4 v2 S0

15. Relation between intensity of fluorescence and concentration
We know that “less number of molecules absorb lesser radiation and so emit lesser radiation”.
“similarly more number of molecules absorb more radiation”.

16. Fluorescence intensity=Q x Ia
Ia -intensity of absorbed light
Q – fluorescence efficiency
Q= fluorescence quanta emitted
EMR quanta absorbed

17. Since Emission is proportional to Absorption.
Ia = Io- It
I0 = Intensity of incident light
It = Intensity of transmitted light
It = I0 e –act
Ia = I0 – I0 e –act
Ia = I0 – (1-e –act)
Ia = I0(1-(1-act)) because e –act =1-act

18. Ia = Io (1-1+act)
Ia = I0 x act
F.I = Q x Ia
F.I = QI0act
Q = constant for a particular substance
I0 = constant for an instrument
a = molecular extinction coefficient
T = path length
C = concentration

19. Fluorescence intensity is proportional to the concentration of the substance.
If the concentration is high it does not fallow due to quenching effect.
concentration
% intensity
Low con
High con

20. Quenching Quenching is the decrease in fluorescence intensity Types :
Self or concentration quenching
Chemical quenching(ph,o2,halides,heavy metals)
Static quenching
Collisional quenching

21. Factors Nature of molecules Nature of substitutent’s
Effect of concentration
Light
Methods of illumination
Oxygen
Photodecomposition
Temperature and viscosity
Adsorption

22. Nature of molecules
Compounds which absorb U.V and VIS radiations shows the fluorescence.
Molecules having conjugated double bond are suitable.
Aliphatic and Saturated cyclic organic compounds are not suitable.

23. Nature of substitutents
Electron donating groups like –NH2, -OH enhance the fluorescence. some of the groups like -SO3H,-NH4 and Alkyl groups not effect Phosphorescence and Fluorescence.
Electron withdrawing groups like -COOH, -NO2,-N=N- and Halides decrease or even destroy the Fluorescence

24. Effect of concentration
Fluorescence α Concentration
(only for small values )
Concentration should low to get good results
weak
strong

25. Light: Monochromatic light is essential.
Methods of illumination :Right angle method is only used ,another method is frontal method.
Oxygen : It shows two effects
- Oxidation of fluorescent substance in to non-fluorescence substance.
- Quenching the fluorescence.
Photodecomposition : Radiation passing through the solutions is weak adequate for the measuring.

26. Photodecomposition : Radiation passing through the solution is weak adequate for the measuring
Temperature and viscosity : variations in temperature and viscosity will cause variations in the frequency
Adsorption: For the fluorimetry we need 10 – 100 weaker solution than the spectrophotometry

27. Instruments
Source : mercury vapor lamp, xenon arc lamp, tungsten lamp.
Filters and monochromators
primary filter
secondary filter
Sample cell
Detectors

28. Instruments Source :Mercury vapor lamp, Xenon arc lamp, Tungsten lamp.
Filters and monochromators
primary filter
secondary filter
Sample cell
Detector’s

29. Single beam fluorimeter
cuvette
Low pass
filter
High pass
filter
Photomultiplier
tube
Tungsten
source

30. Double beam fluorimeter
Sample
Solution
Secondary Filter
Primary Filter
Photo Multiplier Tube
Lamp
Primary Filter
Secondary Filter
Reference
or blank

31. Applications Inorganic chemistry Organic chemistry Biological
Food products
Pharmaceutical
Clinical
Natural products

32. Notes Fluorescence- relaxation through the emission of a photon
Generally occurs on the ms-ns timescale.
Phosphorescence is the emission of a photon from a excited triplet state
Phosphorescence is an extremely rare property

33. Notes Absorption occurs on the 10-15 seconds
Relaxation from the ground state occurs through the fastest available process
Most molecules do not fluoresce because the excited vibrational states of S0 overlap with the S1 state and relaxation can take place rapidly by vibrational relaxation
Generally on the picoseconds timescale

34. Thank you