Flourimetry

Flourimetry- Introduction and Theory. Factors affecting florescence an phosphorescence. Applications of Flourimetry

1. Fluorimetry (Introduction and Theory) Presented by: Atul Adhikari M.Pharm (Pharmaceutics ) Assam down town university, Assam Guided By: Arup Chakraborty Department of Pharmacy, ADTU

2. Introduction Luminescence Fluorescence Fluorophore Fluorimetry Flurometric analysis Fluorescence occurs immediately after the absorption of light and stops as soon as the incident light is cut off. Phosphorescence Phosphorescent substance 2

3. Theory Singlet state: A molecular electronic state in which all the electrons are paired. In a singlet state molecules are diamagnetic. Most of the electrons in ground state are paired. When such molecule absorbs uv/visible radiation, one or more paired electrons reach to the excited Singlet state/excited triplet state. 3

4. Singlet and triplet state The electrons spins in excited state achieved by absorption of radiation may be parallel or antiparallel. 4

5. When radiation is applied with appropriate frequency absorption of light by the molecule cause electrons move from ground state to the excited first singlet state. Once the molecule is in excited state it will try to move to the original state by several ways. These are: Radiationless vibrational deactivation Fluorescence from the excited singlet state Quenching of the excited singlet state Radiationless crossover to the excited triplet state Quenching of the triplet state Phosphorescence form the triplet state 5

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7. Time relationship Of Fluorescence Emission There is considerable time delay between Absorption of light energy Return to the excited state Emission of the fluorescence 7

8. Factors affecting fluorescence and phosphorescence Nature of molecule : Having conjugated double bonds. Temperature/viscosity : Increase in temperature cause decrease in fluorescence. Temperature increase viscosity which in turn decrease fluorescent intensity. Increase in colloision frequency between molecules will increase probability of colloisional deactivation and vibrational relaxation( Colloisional quenching). Temperature of the reaction must be regulated within +/- 0.1 degree centigrade. 8

9. pH In the molecules containing acidic or basic functional groups the change in the medium cause change in ionization of the functional group. Eg: aniline shows fluorescence but it does not show due to formation of anilinium ion in acidic medium. Suitable buffer should be used. 9

10. Dissolved oxygen By direct oxidation of the fluorescent substance to non-fluorescent substance. By quenching of fluorescence. So, useful precaution to check the deaired solution and compare the result obtained with that from the oxygen containing solution should be done. 10

11. Solvent C hanges the polarity or H-bonding ability of the solvent. Different solvent have different ability to stabilize the ground and excited states of the fluorescent molecules. Solvent viscosity and solvents with heavy atoms also affects the fluorescence and phosphorescence. Ethanol can also cause its own fluorescence. 11

12. Concentration effects : Concentration is proportional to the emitted light energy absorbed. At maximum concentration fluorescence peaks and may decrease thereafter. Light effects : Monochromatic light is essential for the excitation of fluoropore because intensity will vary with wavelength. Very long length of exposure cause decrease in fluorescence due to photo decomposition. Increase in intensity of light incident on sample increases fluorescence intensity. 12

13. Adsorption Extreme sensitiveness of the method requires very dilute solutions; 10-100 times weaker than spectrophotometry. Adsorption of substance on container wall is serious problem. E.g. Quinine Certain quartz glass and plastic materials that contain uv adsorbants will fluorescence. 13

14. Nature of substituents : E lectron donating group like -NH2, -OH enhance fluorescence. E lectron withdrawing groups like -COOH, -NO2, -N=N- and hallides destroy fluorescence. If a higher atomic number atom is introduced into a pi-electron system it enhances phosphorescence and decrease fluorescence. 14

15. Applications: Determination of vitamin B2 and B1. Liquid chromatography Organic analysis: quantitative and qualitative analysis of organic aromatic compounds present in cigarette smoke, air pollutants, automobile exhausts, etc. F luorescent indicators: mainly used in acid-base titration e.g: eosin(colorless to green), quinine sulphate(blue to violet). Pharmaceutical analysis. 15

16. Table: Examples of uses of Fluorometry in Pharmaceutical Analysis 16

17. References Willard H., Merritt L, Settle F, Dean J, Instrumental methods of Analysis, 7th edition, New Delhi: CBS publishers and distributors. http:// www.bertholdtech.com Ravi Shankar S, Textbook of Pharmaceutical Analysis. Fourth edition, Tirunelveli: Rx Publications http://en.wikipedia.org/wiki/Fluorescence Gurdeep R Chatwal, Instrumental methods of Chemical analysis 17