Quenching and application of fluorimetry (IMA).pptx

Quenching is the reduction in fluorescence intensity due to interactions with a quencher. It can be dynamic, static, self-quenching, or due to energy transfer (FRET).

Fluorimetry applications include drug analysis, clinical diagnostics, environmental monitoring, and biochemical studies.

1. INSTRUMENTAL METHODS OF ANALYSIS Topic- Quenching and Application of Fluorimetry Prepared by- Pranali V. Lende. B. Pharm 7 th Sem Roll no. 68 Guided by- Ms. S. K. Bhoyar Assist. Professor, Anurag college of pharmacy Warthi, Bhandara.

2. CONTENT Quenching - Introduction - Types of quenching Applications of fluorimetry

3. Quenching - Quenching of fluorescence is physicochemical process which leads to reduction of fluorescence intensity of the sample in the presence of substance other than the fluorescent analyte. The substance which causes quenching is called Quenchers . Quenching may occurs due to the following factors- pH, concentration, temperature, viscosity, presence of O 2 , heavy metals or specific chemical substances.

4. Types of Quenching - Self Quenching / Concentration Quenching Collision Quenching / Dynamic Quenching Chemical Quenching Static Quenching

5. 1. Self Quenching Self quenching is also called as concentration quenching. At low concentration linearity of fluorescence intensity is observed, but as concentration of fluorescing molecule increases in a sample solution the fluorescence intensity decreases.

6. 2. Collision Quenching It also called as Dynamic quenching. It occurs by the collision of a quencher molecule with an excited molecule of the fluorescing substance, leads to reduction in fluorescence intensity. This quenching is occurs when number of collision increased due to- Halides such as chloride, iodides and heavy metals, etc.

7. 3. Chemical Quenching Chemical quenching decrease in fluorescence intensity due to the factors like change in pH , presence of oxygen, halides &heavy metals. p H - Aniline at pH 5-13 gives fluorescence , but at p H <5 &>13 it does not exhibit fluorescence. Halides - like chloride, bromide, iodide & electron withdrawing groups like -NO 2 , -COO H etc. leads to quenching. Heavy metals - leads to quenching, because of collisions of triplet ground state. Oxygen- leads to the oxidation of fluorescence substance to non-fluorescent substance and thus, cause quenching.

8. 4. Static Quenching It is the process in which quencher binds with fluorescent molecule in ground state and inhibits the molecule to go to excited state. Example-Caffeine reduces the fluorescence of riboflavin by complex formation.

9. Application of fluorimetry - 1. Fluorescent indicators - Mainly used in acid-base titration. E.g. Fluorescein: colorless-green
Quinine sulphate: blue-violet Acridine: green-violet 2. Pharmaceutical analysis 3. Determination of vitamin B 1 & B 2

10. 4. Liquid chromatography - Fluorescence is an important method of determining compounds as they appear at the end of chromatogram or capillary electrophoresis column. 5. Organic analysis - Qualitative and quantitative analysis of organic aromatic compounds present in cigarette smoke, air pollutants, automobile exhausts, etc. 6. Determination of inorganic substances - Determination of ruthenium ions in presence of other platinum metals. Determination of aluminum (III) in alloys. Determination of boron in steel by complex formed with benzoin.

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