Spectrofluorometry

1. 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

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. The absorption of light results in the formation of excited molecules which canin turn dissipate their energy by decomposition, reaction, or re-emission. • Theefficiency with which these processes take place is called the quantum efficiencyand 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 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

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