Flourimetry

Theory of flourescence and phosphorescence

1. WELCOME 1

2. THEORY OF FLUORIMETRY PRESENTED BY- AMEENA MEHABOOB 1 ST YR MPHARM 2

3. 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 Phosphorescence Chemiluminescence INTRODUCTION 3

4. FLUORESCENCE When a beam of light is incident on certain substances they emit visible light or radiations. This is called flourescence . It starts immediately after absorption of light and stops as soon as the incident light is cut off. Substances showing this phenomenon are flourescent substances. FLUORESCENCE 4

5. PHOSPHORESCENCE 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 called phosphorescent substances. PHOSPHORESCENCE 5

6. FLOURIMETRY Fluorescence is the molecular absorption of light energy at one wavelength and its nearly instantaneous re-emission at another, usually longer, wavelength. Fluorescent compounds have two characteristic spectra: an excitation spectrum (the wavelength and amount of light absorbed) and an emission spectrum (the wavelength and amount of light emitted). Fluorimetry is the measurement of fluorescence. FLUORIMETRY 6

7. A molecular electronic state in which all the electrons are paired are called singlet state. At this state all molecules are diamagnetic Most of the molecules in their ground state are paired When a molecule absorbs UV/ visible radiation, one or more of the paired electron is raised to an excited singlet state/ excited triplet state Ground singlet state Excited singlet state Spin paired No net mag. field Triplet state Spins unpaired Net mag. field THEORY OF FLUORIMETRY 7

8. SINGLET/TRIPLET STATES Ground state Excited singlet state Excited Triplet state PAULI EXCLUSION PRINCIPLE- No 2 electrons in an atom can have the same set of 4 quantum numbers. In other words, 2 electrons in the same orbital must have opposite spins (paired, no net magnetic field- diamagnetic), molecule with unpaired electrons (triplet state) possess magnetic moment, and are attracted by magnetic field, are called paramagnetic. Excited triplet state is of less energy than excited singlet state. Singlet to triplet transitions are far less probable than singlet/singlet transitions. 8

9. ELECTRONIC AND VIBRATIONAL LEVELS S : ground state of a molecule at ambient temperature S 1 and S 2 : excited singlet states T 1 : lowest energy triplet state, usually of less energy than lowest energetic excited singlet state S 1 Because singlet/triplet transitions are less probable than singlet/singlet transition (spin conversion is necessary), thus average lifetime of an excited triplet state is 10 -4 sec and more, while excited singlet state lifetime is 10 -8 to 10 -5 sec. 9

10. The absorption of a photon of suitable energy causes the molecule to get excited from the ground state to one of the excited states. This process is called as excitation or activation and is governed by Frank‐ Codon principle. According to this principle, the electronic transition takes place so fast (~10‐15s) that the molecule does not get an opportunity to execute a vibration, i.e., when the electrons are excited the inter nuclear distance does not change. The basis for the principle is that the nuclei are very massive as compared to the electrons and therefore move very slowly.  ABSORPTION AND DEACTIVATION 10

11. ELECTRONIC TRANSITIONS 11

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13. The Stokes shift is the gap between the maximum of the first absorption band and the maximum of the fluorescence spectrum STOKES SHIFT heat loss of vibrational energy in the excited state as heat by collision with solvent 13

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15. LIFETIME Excited states decay exponentially with time – I = I e -t/ t I is the initial intensity at time zero, I is the intensity at some later time t t is the lifetime of the excited state. k F = 1/ t , where k F is the rate constant for fluorescence 15

16. QUANTUM YIELD Quantum Yield = F F F F = ratio of photons emitted to photons absorbed Quantum yield is the ratio of photons emitted to photons absorbed by the system: 16

17. Lakowicz , J.R. 1983. Principles of Fluorescence Spectroscopy, Plenum Press, New York. 2 Guilbault , G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York. Instrumental methods of chemical analysis, 3rd Edition, Ewing, G.W., McGraw-Hill Book Co. (1969). REFERENCES 17

18. THANK YOU 18