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  1. 1. SpectrofluorimetryFaculty of Science AZAHARUDDIN M.PHARM, Ph. ANALYSIS F/O PHARMACY JAMIA HAMDARD
  2. 2. FLOURESCENCEFaculty of Science • Molecule absorbs energy and immediately (10-6 to 10- 8 sec) emits energy at a higher wavelength (lower energy) Phosphorescence is similar but involves a slower emission step (> 10-4 sec). • Excitation and emission wavelengths specific to the compound • Emission measured at 90° to the excitation light path • Emission proportional to drug concentration
  3. 3. LUMINESCENCE SPECTROSCOPYFaculty of Science • The emission of radiation from a species after that species has absorbed radiation. FLUORESCENCE LUMINESCENCE PHOSPHORESCENCE SPECTROSCOPY CHEMILUMINESCENCE
  5. 5. LUMINESCENCE SPECTROSCOPYFaculty of Science • In favorable cases, luminescence methods are amongst some of the most sensitive and selective of analytical methods available. • Detection Limits are as a general rule at ppm levels for absorption spectrophotometry and ppb levels for luminescence methods.
  6. 6. LUMINESCENCE SPECTROSCOPYFaculty of Science • Collectively, fluorescence and phosphorescence are known as photoluminescence. • A third type of luminescence - Chemiluminescence - is based upon emission of light from an excited species formed as a result of a chemical reaction.
  7. 7. LUMINESCENCE SPECTROSCOPYFaculty of Science • Most chemical species are not naturally luminescent. • Derivatisation reactions are often available to form luminescent derivatives of non-luminescent compounds. • However, this extra step lessens the attractiveness of luminescence methods.
  9. 9. Fluorescence and Phosphorescence - 1Faculty of Science • Following absorption of radiation, the molecule can lose the absorbed energy by several pathways. The particular pathway followed is governed by the kinetics of several competing reactions.
  10. 10. Fluorescence and Phosphorescence - 2Faculty of Science • One competing process is vibrational relaxation which involves transfer of energy to neighbouring molecules which is very rapid in solution (10-13 sec). – In the gas phase, molecules suffer fewer collisions and it is more common to see the emission of a photon equal in energy to that absorbed in a process known as resonance fluorescence.
  11. 11. Fluorescence and Phosphorescence - 3Faculty of Science • In solution, the molecule rapidly relaxes to the lowest vibrational energy level of the electronic state to which it is excited (in this case S2). The kinetically favoured reaction in solution is then internal conversion which shifts the molecule from S2 to an excited vibrational energy level in S1.
  12. 12. Fluorescence and Phosphorescence - 4Faculty of Science • Following internal conversion, the molecule loses further energy by vibrational relaxation. Because of internal conversion and vibrational relaxation, most molecules in solution will decay to the lowest vibrational energy level of the lowest singlet electronic state before any radiation is emitted.
  13. 13. Fluorescence and Phosphorescence - 5Faculty of Science • When the molecule has reached the lowest vibrational energy level of the lowest singlet electronic energy level then a number of events can take place:
  14. 14. Fluorescence and Phosphorescence - 6Faculty of Science • the molecule can lose energy by internal conversion without loss of a photon of radiation, however, this is the least likely event;
  15. 15. Fluorescence and Phosphorescence - 7Faculty of Science • the molecule can emit a photon of radiation equal in energy to the difference in energy between the singlet electronic level and the ground-state, this is termed fluorescence;
  16. 16. Fluorescence and Phosphorescence - 8Faculty of Science • the molecule can undergo intersystem crossing which involves and electron spin flip from the singlet state into a triplet state. Following this the molecule decays to the lowest vibrational energy level of the triplet state by vibrational relaxation;
  17. 17. Fluorescence and Phosphorescence - 9Faculty of Science • the molecule can then emit a photon of radiation equal to the energy difference between the lowest triplet energy level and the ground-state in a process known as phosphorescence.
  18. 18. Fluorescence and Phosphorescence - 10Faculty of Science • In fluorescence, the lifetime of the molecule in the excited singlet state is 10-9 to 10-7 sec. • In phosphorescence, the lifetime in the excited singlet state is 10-6 to 10 sec (because a transition from T1 to the ground state is spin forbidden).
  19. 19. Quantum EfficiencyFaculty of Science • Fluorescence, phosphorescence and internal conversion are competing processes. The fluorescence quantum efficiency and the phosphorescence quantum efficiency are defined as the fraction of molecules which undergo fluorescence and phosphorescence respectively.
  20. 20. Faculty of Science
  21. 21. Factors affecting Flourescence 1. CONJUGATIONFaculty of Science Molecule must have unsaturation for uv/vis absorption 2. NATURE OF SUBSTITUENT GROUP Electron donating gp-NH2, OH, Increse the FI Electron withdrawing gp-NO2,COOH, Reduce the FI
  22. 22. 3. STRUCTURAL RIGIDITY Rigid structure- More FIFaculty of Science -Flourene Flexile structure-Less FI -Bipheny
  23. 23. Faculty of Science 4.TEMPERATURE High temp reduce the FI due to increase in collision of molecules & vice versa 5.OXYGEN Decrease the FI by oxidation of substance
  24. 24. 6.CONCENTRATION AND FLUORESCENCE INTENSITYFaculty of Science • The power of fluorescent radiation, F, is proportional to the radiant power of the excitation beam absorbed by the species able to undergo fluorescence: F = K(P0 - P) where P0 is the power incident on the sample, P is the power after it traverses a length b of the solution and K is a constant which depends upon experimental factors and the quantum efficiency of fluorescence.
  25. 25. CONCENTRATION AND FLUORESCENCE INTENSITYFaculty of Science • Beers law can be rearranged to give: P/P0 = 10-εbc where A = εbc is the absorbance. Substitution gives: F = KP0(1 - 10- εbc) • This is the fluorescence law • Unlike Beer’s Law fluorescence isn’t in general linear with concentration.
  26. 26. QUENCHING OF FLOURESCENCE • It is decrease in FI due to specific effects of constituents of the solution itself .Faculty of Science • It may be due to con.,PH,Temp,viscosity,presence of specific chemical substances
  27. 27. CONCENTRATION AND FLUORESCENCE INTENSITYFaculty of Science which demonstrates two important points: • that at low concentrations fluorescence intensity is proportional to concentration; • that fluorescence is proportional to the incident power in the incident radiation at the absorption frequency.
  28. 28. INSTRUMENTATION 1. Light sourceFaculty of Science 2. Condencing lens 3. Primary filter 4. Sample container 5. Secondary filter 6. Recorder/Detector
  29. 29. Faculty of Science
  30. 30. 1. Source of light • Mercury vapour lamp –above350 nm at 8 atm p • Xenon arc lamp-more intense radiation thsnFaculty of Science MVP • Tungston lamp-used only for visible region(400- 800nm) 2. Filters & Monochromotors • Primary filter- absorbs vis radiation & transmits uv radiation • Secondory filters- absorbs uv radiation & transmits vis radiation
  31. 31. DETECTORS • Photovoltaic cell • Photo multiplier tubes-most commonFaculty of Science
  32. 32. INSTRUMENTATIONFaculty of Science • The fluorescence is often viewed at 90° orientation (in order to minimise interference from radiation used to excite the fluorescence). • The exciting wavelength is provided by an intense source such as a xenon arc lamp (remember F ∝ P0).
  33. 33. INSTRUMENTATIONFaculty of Science • Because An intense monochromatic light source is required ... • Lasers are an almost ideal light source for fluorimetry (laser-induced fluorescence) but are too expensive and/or impractical for most routine applications. • Two wavelength selectors are required filters (in fluorimeters) and monochromators (in spectrofluorometers).
  34. 34. Types of Fluorescent MoleculesFaculty of Science • Experimentally it is found that fluorescence is favoured in rigid molecules, eg., phenolphthalein and fluorescein are structurally similar as shown below. However, fluorescein shows a far greater fluorescence quantum efficiency because of its rigidity. • phenolphthalein
  35. 35. Types of Fluorescent MoleculesFaculty of Science • It is thought that the extra rigidity imparted by the bridging oxygen group in Fluorescein reduces the rate of nonradiative relaxation so that emission by fluorescence has sufficient time to occur. Fluorescein
  36. 36. APPLICATION 1. Determination of inorganic substances-Faculty of Science e.g. detection of uranium, ruthenium, 2. Determination of organic substances- aromaticpolycylichydrocarbon, indoles, napthols, proteins,pigments, steroids, etc
  37. 37. 3. Pharmaceutical application Aminacrine p-amino salicylateFaculty of Science Desipramine Ergometrine Morphine Indomethacin
  38. 38. APPLICATIONSFaculty of Science B. Fluorimetric Drug Analysis • Many drugs possess high quantum efficiency for fluorescence. For example, quinine can be detected at levels below 1 ppb. Quinine
  39. 39. APPLICATIONSFaculty of Science • In addition to ethical drugs such as quinine, many drugs of abuse fluoresce directly. For example lysergic acid diethylamide (LSD) whose structure is:
  40. 40. Faculty of Science THANK YOU