Quenching of Fluorescence

a substance can absorb any visible light or external radiation and then again emit it. this called fluorescence and the process of reduction in fluorescence intensity is called quenching. this presentation is all about quenching of fluorescence.

1. Quenching of Fluorescence Fatema Tuz Zohora 22 nd batch, Department of Pharmacy, University of Science and Technology Chittagong (USTC).

2. F luorescence Emission of visible light by a substance after absorbing any external radiation is known as fluorescence. There are many drugs and other chemical compounds that show fluorescence property. Such as, riboflavin, vitamin-A, aniline, quinine, fluorescein etc. Fig: Saturated s olution of 0.025g riboflavin in 200 ml water after UV exposure.

3. Fluorometry T he intensity and composition of emitted radiation can be measured by a sensitive method of analysis which is called fluorometry. This method is significant in : Analysis of many drug substances like riboflavin, thiamine, reserpine, quinine etc. Analysis of trace amount of drugs, metabolites, proteins, hormones, steroids etc. in tissues and fluids. Fluorometry is also being used in the treatment and diagnosis of cancer in recent years.

4. Quenching of fluorescence Quenching refers to any process that reduces the fluorescence intensity of a given substance. This may occur due to various factors like pH, concentration, temperature, viscosity, presence of oxygen, heavy metals or, specific chemical substances etc. Fig: Quenching of quinine fluorescence in presence of chloride ions

5. Example of quenching agents Quenching agents Typical fluorophores Thiocyanate Anthracene Chloride Quinine Iodide Tryptophan Disulfide Tyrosine Nitric oxide Naphthalene

6. Fig: T ypes of quenching process Quenching Collisional quenching Static quenching Concentration quenching Chemical quenching

7. Collisional quenching Collisional quenching occurs by the interaction of a quencher molecule ( Q ) with an excited molecule of the fluorescing substance ( F* ). A simplified mechanism can be written to describe this process: F + hv F * F* F + hv F * + Q F + Q* Here, the interaction results in the dissipation of excitation energy by a non radiative energy transfer from F * to Q without or, less fluorescence.

8. Fig : Simple mechanism of collisional quenching Halides ions such as chlorides or, iodides are well known collisional quenchers. For example, quenching of quinine drug by chloride ion or, quenching of tryptophan by iodide ion follow collisional quenching process. Weak coupling L ight Energy transfer Quenching of light F* Q Distance

9. Static quenching Static quenching occurs at the ground state of fluorescing molecule. It can be simplified by following mechanism: F + Q F : Q F:Q + hv Q* + F Q* Q + energy Here, a complex formation occurs between the fluorescing molecule at the ground state ( F ) and the quencher molecule ( Q ) through a strong coupling. Such complex may not undergo excitation or, may be excited to a little extent reducing the fluorescence intensity of the molecule.

10. Strong coupling Fig: S tatic quenching Caffeine and related xanthines and purines reduce intensity of riboflavin by static mechanism. Quenching that occurs due to oxygen also follows this mechanism. Light Energy transfer F Q Quenching of light

11. Concentration quenching Concentration quenching is a kind of self quenching. It occurs when the concentration of the fluorescing molecule increases in a sample solution. The fluorescence intensity is reduced in highly concentrated solution ( >50 μ g/ml ). 100 200 300 concentration (µg/ml) Fig: Influence of concentration on the fluorescence of phenol solutions. 80 60 40 20 f luorescence intensity

12. Chemical quenching Chemical quenching is due to various factors like change in pH, presence of oxygen, halides and electron withdrawing groups, heavy metals etc. Change in pH : Aniline at pH (5-13) gives fluorescence when excited at 290 nm. But pH <5 or, pH >13 it does not show any fluorescence . Oxygen : Oxygen leads to the oxidation of fluorescent substance to non fluorescent substance and thus, causes quenching.

13. Halides and electron withdrawing groups : Halides like chloride ions, iodide ions and electron withdrawing groups like -NO , -COOH , -CHO groups lead to quenching. Heavy metals : presence of heavy metals also lead to quenching because of collision and complex formation. 2 Fig: e lectron withdrawal process by nitro group

14. Conclusion In the usual case, quenching is an undesirable effect and the possibility of encountering this type of interference should always be evaluated in developing a fluorometric assay. However, this phenomenon can be used as an analytical means for determining the concentration of the compounds known to quench fluorescence. Quenching study can also be used to reveal the localization of fluorophores in proteins or, membranes and their permeability to the quenchers.

15. References Leslie G. Chatten , Pharmaceutical Chemistry T heory and Application (volume-2), CBS publishers and distributors, 2014 revised edition, page: 180,181,182,183. Joseph R. Lakowicz, Principles of Fluorescence Spectroscopy, 3 rd edition (2006), Springer Science publication, page: 278,282,283. Wikipedia//Quenching(Fluorescence).

16. Thank you…