1. For B.Pharm IIIrd yr students
Spectrofluorometry
For B.Pharm IIIrd yr students

2. Definitions
Incandescence is the emission of electromagnetic radiation from a hot body as a result of its temperature.
EX. Incandescence is exploited in incandescent light bulbs, in which a filament is heated to a temperature at which a fraction of the radiation falls in the visible spectrum.
All other forms of light emission are called Luminescence
Luminescence is emission of light by a substance not resulting from heat; it is thus a form of cold-body radiation. It can be caused by chemical reactions, electrical energy, subatomic motions, or stress on a crystal, which all are ultimately caused by Spontaneous emission.
_______ as in fluorescence and phosphorescence.

3. Fluoroscence
Fluoroscence is the emission of light by a substance that has absorbed EMR.
Fluorophores can absorb/emit light of different wavelength
Fluorescence spectrum
Absorption/excitation spectrum
Emission spectrum

4. Why is the emission spectra at longer wavelength????
Stoke’s law
Internal conversion
Red shift
We will get to this later!

5. Spectrofluorometry
Spectrofluorometry is commonly defined as a type of spectrophotometry which deals with fluorescence, the phenomenon in which light incident on a material at a given wavelength, usually in the ultraviolet or visible spectral range, causes that material to radiate light at longer, less energetic wavelengths.

6. ФE = No. of quanta emitted No. of quanta absorbed
The absorption of light results in the formation of excited molecules which can in turn dissipate their energy by decomposition, reaction, or re-emission.
The efficiency with which these processes take place is called the quantum efficiency and in the case of photoluminescence can be defined as:
ФE = No. of quanta emitted No. of quanta absorbed
and never exceeds unity

7. What wavelength of light must be shone to excite the electrons?????
Jablonski diagram

8. Remember this?

11. Possible ways of relaxation How the e-moves ……
Vibrational relaxation
Internal conversion (goes to lower vibrational level)
Intersystem crossing (goes to the triplet excited state)
Fluorescence
Delayed fluorescence (interconversion + intersystem crossing to the triplet exc. State – back and forth)
Phosphorescence (time lag: intersystem crossing from triplet excited state)

12. Intersystem crossing/heavy atom effect
"forbidden" spin transition
When an electron in a molecule with a singlet ground state is excited (via absorption of radiation) to a higher energy level, either an excited singlet state or an excited triplet state will form. A singlet state is a molecular electronic state such that all electron spins are paired. That is, the spin of the excited electron is still paired with the ground state electron (a pair of electrons in the same energy level must have opposite spins, per the Pauli exclusion principle). In a triplet state the excited electron is no longer paired with the ground state electron; that is, they are parallel (same spin). Since excitation to a triplet state involves an additional "forbidden" spin transition, it is less probable that a triplet state will form when the molecule absorbs radiation.
Singlet and triplet energy levels.
When a singlet state nonradiatively passes to a triplet state, or conversely a triplet transitions to a singlet, that process is known as intersystem crossing
Excited electrons can undergo intersystem crossing to a degenerate state with a different spin multiplicity.
Can lead to decrease in fluorescence
And cause phosphorescnce

13. Figure showing Fluorescence and Phosphorescence

14. Fluorescence lifetime
The characteristic time that the fluorophore spends in the excited state.
During the time the e- spends in the excited state the fluorophore undergoes multiple interactions with the environment.
Collision quenching
Energy transfer
Intersystem crossing
Rotational motion

15. Fluorescence yiel/quantum yield and factors affecting it
Ø = kf
kf+ki+Kec+kic+kpd+kd
if, kx = ki+Kec+kic+kpd+kd
Ø = kf
kf+ kx
K is the respective rate constants for the various deactivation processes

16. Variables affecting fluorescence
Fluorescence and structure
Structural rigidity
Effect of temperature and solvent
Effect of pH
Effect of concentration on intensity

17. Effect of structure on Fluo.
Simple Heterocycles do not exhibit fluorescence
Pyridine
furan
thiophene
pyrrole
The quantum energy increases with increase in number of rings and their degree of condensation
Isoquinoline
quinoline
indole
Condensation and substitution of heterocyclics causes fluorescence

18. Influence of substitution
Influence of a halogen substitution decreases fluorescence as the molar mass of the halogen increases.  This is an example of  the “heavy atom effect”  which suggest that the probability of intersystem crossing increases as the size of the molecule increases. 
Fluorescence decreases

19. Table: Relative intensity fluorescence comparison with halogen substituted compounds
Wavelength nm
Flu. intensity
Fluorobenzene 10
Chlorobenzene 7
Bromobenzene 5

20. No fluorescence
In heavy atom substitution such as nitro derivatives or heavy halogen substitution such as iodobenzene, the compounds are subject to predissociation. These compounds have bonds that easily rupture that can then absorb excitation energy and go through internal conversion.
Iodobenzene and nitro benzene do not show fluorescence

21. Effect of structure rigidity
Fluorescence is particularly favored in molecules with rigid structures.  The table below compares the quantum efficiencies of fluorine and biphenyl which are both similar in structure that there is a bond between the two benzene group.  The difference is that fluorene is more rigid from the addition methylene bridging group. 
Fluorescence is favored in rigid molecules.
QE 0.2
QE 1

22. Fluorescence of organic chelating agent when the compound is complexed with a metal ion.
 8-hydroxyquinoline with Zinc complexed
8-hydroxyquinoline     
 higher fluorescence intensity

23. THANK YOU