Flourimetry
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The document discusses fluorimetry, an analytical method based on the measurement of fluorescence emitted by molecules after absorbing electromagnetic radiation. It covers the principles of fluorescence, types of fluorescence, factors affecting intensity, quenching mechanisms, and instrumentation used in fluorimetry. Additionally, it highlights various applications of fluorimetry in determining organic and inorganic compounds.
Flourimetry
- 1. FLUORIMETRY AFSATH.B M PHARM 1ST YEAR PHARMACOGNOSY AND PHYTOCHEMISTRY MALIK DEENAR COLLEGE OF PHARMACY
- 2. • SPECTROSCPY : Study of interaction of EMR with matter, which may result in absorption, transmission, emission,reflection , rotation of EMR • Luminescence is the phenomenon of emission of photons (light radiationbs) by a substance when excitation occur bybany form • 3 types • Chemiluminescence spectroscopy • Electro-Chemiluminescence spectroscopy • Photoluminescence • FLUORIMETRY : Ananalytical method for the measurement of fluorescence, based upon emission of absorbed radiation by the molecules. When a molecule absorbs incident electromagnetic radiations, it is excited to higher energy level,where it is unstable. • Study of measurement of emitted radiations (ie from singlet ES to singlet GS)- Fluorimetry • Study of measurement of emitted radiations when electrons undergotransition from TS
- 3. THEORY OF FLUORESCENCE & PHOSPHORESCENCE • A molecular electronic state in which all of the electrons are paired are called singlet state. • Most of the molecule in their ground state are paired. • When such molecule absorb uv/visible radiation , one or more of the paired electron raised to an excited singlet state/ excited triplet state. TYPES OF FLUORESCENCE a. Based on wave length of emitted radiation • Stokes fluorescence(Emitted radiation – longer than the absorbed radiation) • Anti stoke fluorescence(Emitted radiation –shorter than the absorbed radiation) • Resonance fluorescence(Emitted radiation – equal to the absorbed radiation) b. Based on phenomenon • Sensitized fluorescence • Direct line fluorescence • Stepwise fluorescence • Thermally assisted fluorescence
- 5. FACTORS AFFECTING FLUORESCENCE INTENSITY •Nature of substituents •Rigidity of structure •Concentration •Oxygen •pH •Photodecomposition •Temperature •Viscosity
- 6. 1. CONJUGATION Molecule must have unsaturation-Absorb radiations-fluoresces 2. NATURE OF SUBSTITUENTS • Electron donating group likeNH2, OH…enhances fluorescence intensity • Electron withdrawing group like NO2, COOH…reduces FI 3. RIGIDITY OF STRUCTURE • Rigid structure more FI Flexible structure less FI
- 7. 4. TEMPERATURE increase temp lead to increase collision of moleculedecrease FI decrease temp lead to decrease collision of molecule increase FI 5. VISCOSITY inversely proportional to effect of temperature 6. OXYGEN decreases FI in 2 ways; • Oxidises fluorescent substances to non-fluorescent substances • And quenches FI because of paramagnetic property 7. PH PH depends on chemical structure of molecule
- 8. 8. PHOTODECOMPOSITION Influence of wave length leads to photo chemical decomposition 9. CONCENTRATION less no. of molecule absorb less no. of radiation & vice versa is the true. FI ∝ 1/Conc
- 9. QUENCHING Decrease in FI due to various factors like; conc, Ph , temp, viscosity, presence halides … TYPES 1. SELF/ CONCENTRATION QUENCHING 2. CHEMICAL QUENCHING 3. STATIC QUENCHING 4. COLLISIONAL QUENCHING STATIC QUENCHING This occurs because of complex formation .Eg,caffeine reduces FI of Riboflavin
- 10. COLLISIONAL QUENCHING • Two samples of quinine dissolved in water with a violet laser (left) illuminating both. Typically quinine fluoresces blue, visible in the right sample. The left sample contains chloride ions which quench quinine's fluorescence, so the left sample does not fluoresce visibly (the violet light is just scattered laser light).
- 11. INSTRUMENTATION •Source of light •Filter or mono-chromators •Sample Cell •Detectors.
- 12. MERCURY VAPOUR LAMP XENON ARC LAMP TUNGSTEN LAMP SOURCE OF LIGHT
- 13. FILTERS & MONOCHROMATORS FILTERS • Used in inexpensive instruments • Consider excitation wavelength & emission wavelenghth • 2 filters –primary & secondary • Primary filters – absorb visible radiation & transmit UV rays • Secondary – absorb UV radiation & transmit visible rays MONOCHROMATORS • Used in expensiveflourimeter like spectroflourimeter • 2 types- excitation & emission monochromators • Has gratings
- 14. SAMPLE CELL • Cylindrical or polyhedral • Cells are made up of colour corrected fused glass • Path length -10mm/1 cm • No need of cells made of quartz
- 15. DETECTORS PHOTO VOLTAIC CELL PHOTO TUBES
- 16. PHOTO MUTI[PLIER TUBE (PMT) • Aphoto cathode & series of dynodes are used • Cathodes are maintained at 75-100V
- 17. INSTRUMENTS •SINGLE BEAM FILTER FLUORIMETER •DOUBLE BEAM FILTER FLUORIMETER •SPECTROFLUORIMETER (Double beam )
- 18. SINGLE BEAM FILTER FLUORIMETER • Tunsten lamp as source • Primary secondary filters used • Primary filters – absorb visible radiation & transmit UV rays • Secondary – absorb UV radiation & transmit visible rays • Emitted radiation measured at 90˚ by secondary filter Advantages • Simple in construction • Easy to use • Economical Disadvantages • It is not possible to use reference solution & sample solution at a time • Rapid scanning to obtain Exitation & emission spectrum of the compound is not possible.
- 20. DOUBLE BEAM FILTER FLUORIMETER • Similar to single beam instrument •Two incident beams from light source pass through primary filters separately and fall on either sample or reference solution •The emitted radiation from sample or reference pass separately through secondary filter. •Advantage •Sample & reference solution can be analyzed simultaneously. •Disadvantage •Rapid scanning is not possible due to use of filters.
- 22. SPECTROFLUORIMETER • Working similar to double beam filter flourimetr • Instead of filter monochromators are used
- 23. APPLICATION • Determination of organic • Inorganic compounds –proteins, naphthols, pigments, steroids • Flourescent compounds- griseofulvin, morphine, quinine, riboflavin… • Non fluorescent compounds- heroin , isoniazid, reserpine… • Mixture of medical substances • Determination of aspirin in blood serum. • Estimation of cocaine and atropine in urine.
- 24. REFERENCES • Sharma Y R. Elementary organic spectroscopy . 16th ed . s chand &company Ltd. New delhi 2007. • Gurudeep R chatwal.Sham K Anand.Instrumental method chemical analysis.5th ed.Himalaya publishing house.Mumbai 2002.p.2.399-2.416. • Ravi Sankar .text boook of pharmaceutical analysis.3rd ed. Rx publication.2006. • Wikipidea.org • https://image.slidesharecdn.com/fluorimetryphosphorimetry • https://www.google.co.in/search