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By
K. RAKESH GUPTA
 INTRODUCTION
 DEFINITION
 THEORY
 FACTORS AFFECTING FLOURESCENCE
 INSTRUMENTATION
 APPLICATIONS IN PHARMACY
 CONCL...
Luminescence is the emission of light by a
substance. It occurs when an electron
returns to the electronic ground state fr...
When a beam of light is incident on certain
substances they emit visible light or
radiations. This is known as fluorescenc...
 When light radiation is incident on certain
substances they emit light continuously even
after the incident light is cut...
 A molecular electronic state in which all of the
electrons are paired are called singlet state.
 In a singlet state mol...
Ground excited singlet triplet state
singlet state spins unpaired
states spin paired
no net mag.field net mag.field
Fluorescence
Phosphorescence
Radiation less processes
Vibration relaxation
Internal conversion
External conversion
...
LIGHT EMITING AT ONCE SOURCE STARTS &
STOPS WHEM SOURCE STOPS
JABLONSKI ENERGY DIAGRAM
FLUORESCENCE AND
CHEMICAL STRUCTURE
Fluorescence is most commonly observed in
compounds containing aromatic functional
gr...
CONTD…
Simple heterocyclic do not exhibit
fluorescence.
The n - *singlet quickly converts to the
n - * triplet and pre...
Fusion of heterocyclic nucleus to benzene ring
increases fluorescence.
Substitution on the benzene ring shifts
wavelength of absorbance maxima and
corresponding changes in fluorescence
peaks
...
 Fluorescence is favored in molecules
with structural rigidity.
 organic chelating agents complexed with
metal ion incre...
 Nature of molecule
 Nature of substituent
 Effect of concentration
 Adsorption, Light
 Oxygen,ph
 Photodecompositio...
nature of molecules
All the molecules cannot show the
phenomenon of fluorescence.
Only the molecules absorbs uv/visible
...
nature of substituent
Electron donating group enhances
fluorescence – e.g.:NH2,OH etc.
Electron withdrawing groups decre...
Fluorescence is directly
proportional to concentration.
FI = Q X Ia
i.e, F = QIOact
Q = Constant for a particular substance
IO = Constant for an instrument
a = Molecular extincti...
 Extreme sensitiveness of the method
requires very dilute solution.
Adsorption of the fluorescent substances on
the cont...
 Monochromatic light is essential for the
excitation of fluorescence because the
intensity will vary with wavelength.
OXY...
 Alteration of the ph of the solution will have
significant effect on fluorescence.
 Fluorescent spectrum is different f...
Kf = fluorescence
kec = external conversion
kic = internal conversion
kisc = intersystem crossing
kpd = pre dissociation
K...
 Increase in intensity of light incident on
sample increases fluorescence intensity.
 The intensity of light depends upo...
 The effective path length depends on
both the excitation and emission slit
width.
 Use of microcuvette does not reduce
...
 Decrease in fluorescence intensity due to specific
effects of constituents of the solution.
 Due to concentration, ph, ...
Fluorescence
Concentration of
fluorescing species
Deviations at higher concentrations can be
attributed to self-quenching ...
 Here decrease in fluorescence intensity due to
the factors like change in ph,presence of
oxygen, halides &heavy metals.
...
 This occurs due to complex formation.
e.g.. caffeine reduces the fluorescence of
riboflavin by complex formation.
COLLIS...
INSTRUMENTATION
 SOURCE OF LIGHT
 FILTERS AND MONOCHROMATORS
 SAMPLE CELLS
 DETECTORS
 MERCURY ARC LAMP.
 XENON ARC LAMP.
 TUNGSTEN LAMP.
 TUNABLE DYE LASERS.
MERCURY ARC LAMP
Produce intense line spectrum above 350nm.
High pressure lamps give lines at 366,405, 436,
546,577,691,...
 Intense radiation by passage of current through an
atmosphere of xenon.
 Spectrum is continuous over the range between ...
Intensity of the lamp is low.
If excitation is done in the visible
region this lamp is used.
It does not offer UV radia...
Pulsed nitrogen laser as the
primary source.
Radiation in the range between
360 and 650 nm is produced.
FILTERS
Primary filter-absorbs visible light & transmits
uv light.
Secondary filter-absorbs uv radiations &
transmits visi...
 The majority of fluorescence assays are carried out in
solution.
 Cylindrical or rectangular cells fabricated of silica...
PHOTOVOLTAIC CELL
PHOTO TUBE
PHOTOMULTIPLIER TUBES – Best
and accurate.
Multiplication of photo electrons by
secondary emission of radiation.
A photo cathode and series of dynodes are
used.
E...
Tungsten lamp as source of light.
The primary filter absorbs visible radiation
and transmits uv radiation.
Emitted radi...
 Simple in construction
 Easy to use.
 Economical
disadvantages
 It is not possible to use reference solution &
sample...
 Similar to single beam instrument.
 Two incident beams from light source pass through
primary filters separately and fa...
 Sample & reference solution can be analyzed
simultaneously.
disadvantage
 Rapid scanning is not possible due to use of
...
Power
supply
Source primary filter
secondary filter
Detector
Sample cell
Slit
Data processor
1] Determination of inorganic substances
Determination of ruthenium ions in presence of
other platinum metals.
Determina...
 Field determination of uranium salts.
3]fluorescent indicators
Mainly used in acid-base titration.
e.g.:
eosin- colorles...
Reagent Ion Fluorescence
wavelength
Sensitivity
Alizarin
garnet B
Al3+ 500 0.007
Flavanol
8-Hydroxy
quinoline
Sn4+
Li2+
47...
compound reagent excitation
wavelength
fluorescence
hydrocortisone 75%v/v
H2SO4 in
ethanol
460 520
nicotinamide cyanogen
c...
7] Liquid chromatography
Fluorescence is an imp method of
determining compounds as they
appear at the end of chromatogram...
Douglas A Skoog, Principles of instrumental
analysis
 H:UV-Vis Luminescence Spectroscopy - Theory.mht
Dr.B.K.Sharma, In...
 http://images.google.co.in/imghp?oe=UTF-
8&hl=en&tab=wi&q=fluorescence
 http://en.wikipedia.org/wiki/Fluorescence
 htt...
Fluorimetry
Fluorimetry
Fluorimetry
Fluorimetry
Fluorimetry
Fluorimetry
Fluorimetry
Fluorimetry
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Fluorimetry

  1. 1. By K. RAKESH GUPTA
  2. 2.  INTRODUCTION  DEFINITION  THEORY  FACTORS AFFECTING FLOURESCENCE  INSTRUMENTATION  APPLICATIONS IN PHARMACY  CONCLUSION  REFERENCES
  3. 3. Luminescence is the emission of light by a substance. It occurs when an electron returns to the electronic ground state from an excited state and loses its excess energy as a photon. It is of 3 types. Fluorescence spectroscopy. Phosphorescence spectroscopy. Chemiluminescence spectroscopy
  4. 4. When a beam of light is incident on certain substances they emit visible light or radiations. This is known as fluorescence. Fluorescence starts immediately after the absorption of light and stops as soon as the incident light is cut off. The substances showing this phenomenon are known as flourescent substances.
  5. 5.  When light radiation is incident on certain substances they emit light continuously even after the incident light is cut off.  This type of delayed fluorescence is called phosphorescence.  Substances showing phosphorescence are phosphorescent substances.
  6. 6.  A molecular electronic state in which all of the electrons are paired are called singlet state.  In a singlet state molecules are diamagnetic.  Most of the molecules in their ground state are paired.  When such a molecule absorbs uv/visible radiation, one or more of the paired electron raised to an excited singlet state /excited triplet state.
  7. 7. Ground excited singlet triplet state singlet state spins unpaired states spin paired no net mag.field net mag.field
  8. 8. Fluorescence Phosphorescence Radiation less processes Vibration relaxation Internal conversion External conversion Intersystem crossing
  9. 9. LIGHT EMITING AT ONCE SOURCE STARTS & STOPS WHEM SOURCE STOPS
  10. 10. JABLONSKI ENERGY DIAGRAM
  11. 11. FLUORESCENCE AND CHEMICAL STRUCTURE Fluorescence is most commonly observed in compounds containing aromatic functional groups with low energy. Most unsubstituted aromatic hydrocarbons show fluorescence - quantum efficiency increases with the no: of rings and degree of condensation.
  12. 12. CONTD… Simple heterocyclic do not exhibit fluorescence. The n - *singlet quickly converts to the n - * triplet and prevents fluorescence.
  13. 13. Fusion of heterocyclic nucleus to benzene ring increases fluorescence.
  14. 14. Substitution on the benzene ring shifts wavelength of absorbance maxima and corresponding changes in fluorescence peaks  Fluorescence decreases with increasing atomic no: of the halogen.  Substitution of carboxylic acid or carboxylic group on aromatic ring inhibits fluorescence.
  15. 15.  Fluorescence is favored in molecules with structural rigidity.  organic chelating agents complexed with metal ion increases fluorescence.
  16. 16.  Nature of molecule  Nature of substituent  Effect of concentration  Adsorption, Light  Oxygen,ph  Photodecomposition  Temp . &viscosity  Quantum yield  Intensity of incident light  Path length
  17. 17. nature of molecules All the molecules cannot show the phenomenon of fluorescence. Only the molecules absorbs uv/visible radiation can show this phenomenon. Greater the absorbency of the molecule the more intense its fluorescence.
  18. 18. nature of substituent Electron donating group enhances fluorescence – e.g.:NH2,OH etc. Electron withdrawing groups decrease or destroy fluorescence. e.g.:COOH,NO2, N=N etc. High atomic no: atom introduced into  electron system decreases fluorescence.
  19. 19. Fluorescence is directly proportional to concentration.
  20. 20. FI = Q X Ia i.e, F = QIOact Q = Constant for a particular substance IO = Constant for an instrument a = Molecular extinction coefficient t = Path length C = Concentration of the substance F = KC Where K represents all constants FI α Concentration.
  21. 21.  Extreme sensitiveness of the method requires very dilute solution. Adsorption of the fluorescent substances on the container wall create serious problems. Hence strong solutions must be diluted.
  22. 22.  Monochromatic light is essential for the excitation of fluorescence because the intensity will vary with wavelength. OXYGEN The presence of oxygen may interfere in 2 ways. 1] by direct oxidation of the fluorescent substances to non fluorescent. 2] by quenching of fluorescence.
  23. 23.  Alteration of the ph of the solution will have significant effect on fluorescence.  Fluorescent spectrum is different for ionized and un-ionized species. TEMPERATURE & VISCOSITY  Increase in temperature/decrease in viscosity will decrease fluorescence.
  24. 24. Kf = fluorescence kec = external conversion kic = internal conversion kisc = intersystem crossing kpd = pre dissociation Kd = dissociation fluorescence quantum yield:
  25. 25.  Increase in intensity of light incident on sample increases fluorescence intensity.  The intensity of light depends upon 1)light emitted from the lamp. 2)Excitation monochromaters 3)Excitation slit width
  26. 26.  The effective path length depends on both the excitation and emission slit width.  Use of microcuvette does not reduce the fluorescence.  Use of microcell may reduce interferences and increases the measured fluorescence
  27. 27.  Decrease in fluorescence intensity due to specific effects of constituents of the solution.  Due to concentration, ph, pressure of chemical substances, temperature, viscosity, etc. Types of quenching Self quenching Chemical quenching Static quenching Collision quenching
  28. 28. Fluorescence Concentration of fluorescing species Deviations at higher concentrations can be attributed to self-quenching or self-absorption. Fluorescence Concentration of fluorescing species Calibration curve (Low con) calibration curve (High con)
  29. 29.  Here decrease in fluorescence intensity due to the factors like change in ph,presence of oxygen, halides &heavy metals.  ph- aniline at ph 5-13 gives fluorescence but at ph <5 &>13 it does not exhibit fluorescence.  halides like chloride,bromide,iodide & electron withdrawing groups like no2,cooH etc. leads to quenching.  Heavy metals leads to quenching, because of collisions of triplet ground state.
  30. 30.  This occurs due to complex formation. e.g.. caffeine reduces the fluorescence of riboflavin by complex formation. COLLISIONAL QUENCHING  It reduces fluorescence by collision. where no. of collisions increased hence quenching takes place.
  31. 31. INSTRUMENTATION
  32. 32.  SOURCE OF LIGHT  FILTERS AND MONOCHROMATORS  SAMPLE CELLS  DETECTORS
  33. 33.  MERCURY ARC LAMP.  XENON ARC LAMP.  TUNGSTEN LAMP.  TUNABLE DYE LASERS.
  34. 34. MERCURY ARC LAMP Produce intense line spectrum above 350nm. High pressure lamps give lines at 366,405, 436, 546,577,691,734nm. Low pressure lamps give additional radiation at 254nm.
  35. 35.  Intense radiation by passage of current through an atmosphere of xenon.  Spectrum is continuous over the range between over 250- 600nm,peak intensity about 470nm.
  36. 36. Intensity of the lamp is low. If excitation is done in the visible region this lamp is used. It does not offer UV radiation.
  37. 37. Pulsed nitrogen laser as the primary source. Radiation in the range between 360 and 650 nm is produced.
  38. 38. FILTERS Primary filter-absorbs visible light & transmits uv light. Secondary filter-absorbs uv radiations & transmits visible light. MONOCHROMATORS Exitation monochromaters-isolates only the radiation which is absorbed by the molecule. Emission monochromaters-isolates only the radiation emitted by the molecule.
  39. 39.  The majority of fluorescence assays are carried out in solution.  Cylindrical or rectangular cells fabricated of silica or glass used.  Path length is usually 10mm or 1cm.  All the surfaces of the sample holder are polished in fluorimetry.
  40. 40. PHOTOVOLTAIC CELL PHOTO TUBE PHOTOMULTIPLIER TUBES – Best and accurate.
  41. 41. Multiplication of photo electrons by secondary emission of radiation. A photo cathode and series of dynodes are used. Each cathode is maintained at 75-100v higher than the preceding one. Over all amplification of 106 is obtained.
  42. 42. Tungsten lamp as source of light. The primary filter absorbs visible radiation and transmits uv radiation. Emitted radiation measured at 90o by secondary filter. Secondary filter absorbs uv radiation and transmits visible radiation.
  43. 43.  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.
  44. 44.  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.
  45. 45.  Sample & reference solution can be analyzed simultaneously. disadvantage  Rapid scanning is not possible due to use of filters.
  46. 46. Power supply Source primary filter secondary filter Detector Sample cell Slit Data processor
  47. 47. 1] 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. Estimation of cadmium with 2-(2 hydroxyphenyl) benzoxazole in presence of tartarate.
  48. 48.  Field determination of uranium salts. 3]fluorescent indicators Mainly used in acid-base titration. e.g.: eosin- colorless-green. Fluorescein:colourless-green. Quinine sulphate: blue-violet. Acridine: green-violet
  49. 49. Reagent Ion Fluorescence wavelength Sensitivity Alizarin garnet B Al3+ 500 0.007 Flavanol 8-Hydroxy quinoline Sn4+ Li2+ 470 580 0.1 0.2 4] Fluorometric reagent  Aromatic structure with two or more donor functional groups
  50. 50. compound reagent excitation wavelength fluorescence hydrocortisone 75%v/v H2SO4 in ethanol 460 520 nicotinamide cyanogen chloride 250 430 5] organic analysis Qualitative and quantitative analysis of organic aromatic compounds present in cigarette smoke, air pollutants, automobile exhausts etc. 6] pharmaceutical analysis
  51. 51. 7] Liquid chromatography Fluorescence is an imp method of determining compounds as they appear at the end of chromatogram or capillary electrophoresis column. 8]determination of vitamin B1 &B2.
  52. 52. Douglas A Skoog, Principles of instrumental analysis  H:UV-Vis Luminescence Spectroscopy - Theory.mht Dr.B.K.Sharma, Instrumental methods of chemical analysis Gurdeep R Chatwal, Instrumental methods of chemical analysis
  53. 53.  http://images.google.co.in/imghp?oe=UTF- 8&hl=en&tab=wi&q=fluorescence  http://en.wikipedia.org/wiki/Fluorescence  http://www.bertholdtech.com/ww/en pub/bioanalytik/biomethods/fluor.cfm
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