This document discusses various factors that affect fluorescence intensity, which is determined by the emission of light from fluorophores following energy absorption. Key factors include the molecular structure, substituent groups, temperature, viscosity, oxygen presence, pH, concentration, and quenching effects. The measurement of fluorescence intensity is crucial for identifying fluorophores and their concentrations in biochemical assays.

1. FACTORS AFFECTING
FLUORESCENCE INTENSITY
PRESENTED BY,
BHUVANESWARAN D
M.PHARM-1ST SEMESTER
DEPT.OF PHARM.ANALYSIS
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2. FLUORESCENCE
 Fluorescence is based on photoluminescence, a process
of glow and light emission. It is a physical process in which
light is emitted after it has been absorbed by a substance.
The fluorescence intensity indicates how much light
(photons) is emitted.
 Fluorescence is created by the absorption of energy (light)
by fluorescent molecules, called fluorophores.
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3. JABLONSKI DIAGRAM
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4. FACTORS AFFECTING FLUORESCENCE
INTENSITY
 Conjugation
 Nature of substituent group
 Rigidity of structure
 Effect of temperature
 Viscosity
 Oxygen
 Effect of pH
 Photochemical decomposition
 Effect of concentration
 Quenching
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5. I. CONJUGATION
 A molecule must have an unsaturation (π electrons i.e.
Conjugation)
 So that molecule absorbs UV/Visible radiation, which leads
to increase in fluorescence.
 If there is no absorption of radiation, there will not be any
fluorescence.
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6. II. NATURE OF SUBSTITUENT GROUPS
 Electron donating groups like amino (NH2) and hydroxyl
(OH) groups increasing the fluorescence intensity.
 Electron withdrawing groups like nitro (NO2) and carboxyl
(COOH) groups decreasing the fluorescence intensity.
 Groups like SO3 or NH4 have no effect on fluorescence
intensity.
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7. S.NO SUBSTITUENTS EFFECT ON λ EFFECT ON INTENSITY
1. Alkyl No effect Slight increase or decrease
2. COOH, CHO, COOR, COR Increase Decrease
3. OH, OMe, Oet Increase Increase
4. CN No effect Increase
5. NH2, NHR, NR Increase Increase
6. NO2, NO Increase Increase
7. SH Increase Decrease
8. SO3H No effect No effect
9. F, CL, Br, I Increase Decrease
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8. Electron donating group Electron withdrawing group
Ed
Phenol Benzene Benzoic acid
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9. III. RIGIDITY OF STRUCTURE
 Rigid structure gives more
fluorescence intensity.
 Flexible structure gives less
fluorescence intensity.
E.g. : Fluorene
E.g. : Biphenyl
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10. IV. EFFECT OF TEMPERATURE
 Increase in temperature leads to increase in
collision of molecules.
Fluorescence intensity
Decrease in temperature leads to decrease
in collision of molecules.
Fluorescence intensity
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11. V. VISCOSITY
 Increase in viscosity leads to
decrease in collision of molecules.
Fluorescence intensity
 Decrease in viscosity leads to
increase in collision of molecules.
Fluorescence intensity
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12. VI. OXYGEN
 Oxygen decreases the fluorescence intensity in two ways:
i. It oxidizes fluorescent substance to non-fluorescent
substance.
ii. It quenches (decrease) fluorescence, because of the
paramagnetic properties of molecular energy, as it has
triplet ground state.
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13. VII. EFFECT OF pH
 The effect of pH depends on the chemical structure of molecule.
i. Phenols in acidic condition are undissociated and do not give
fluorescence, but in alkaline condition, they are dissociated
(ionic) and gives good fluorescent.
Phenol phenoxide ion
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14. ii. Aniline in neutral or alkaline gives visible fluorescence but in acidic
condition gives fluorescence in UV region only.
Aniline Anilinium ion
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15. VIII.PHOTOCHEMICAL DECOMPOSITION
UV/vis absorption sometimes leads to photochemical reaction. In
such case, fluorescence cannot be seen. Hence a wavelength which
is not strongly absorbed should be chosen to avoid such a reaction.
Otherwise errors up to 20% is possible.
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16. IX. EFFECT OF CONCENTRATION
Fluorescence intensity is directly proportional to the concentration
of the substances.
i. At high concentration
seeing deviation in
linearity. i.e. When
concentration increases,
fluorescence intensity
does not increase.
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17. At low concentration
seeing no deviation in
linearity. i.e. When
concentration increases,
fluorescence intensity
increase.
ii.
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18. X. QUENCHING
 Quenching is the decrease in fluorescence intensity due to specific
effects of constituents of solution itself.
 Various types of quenching are,
1) Self quenching
2) Chemical quenching
3) Static quenching
4) Collision quenching
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19. i. SELF QUENCHING
At low concentration linearity should be observed, at high
concentration of same substance, increase in fluorescence
intensity does not occur. This phenomenon is called as self
quenching or concentration quenching.
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20. ii. CHEMICAL QUENCHING
 It occurs due to the following factors,
Change in pH
Presence of oxygen
Halides or heavy metals
1. pH: -
Aniline at pH 5 to 13 gives fluorescence when excited at
290nm. But at < 5 (exist as cation) and pH at >13 (exist as anion)
it does not exhibit fluorescence.
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21. 2. OXYGEN: -
Prescence of oxygen leads to quenching because of its paramagnetic
property (triplet ground state).
3. HALIDES: -
Halides like bromide, chloride, iodide and electron withdrawing group
like nitro and carboxylic group leads to quenching. (decrease in
fluorescence intensity)
4. HEAVY METALS: -
Prescence of heavy metals also leads to quenching because of its
collision and triplet ground state.
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22. iii. STATIC QUENCHING: -
 This occurs because of its complex formation. (i.e. caffeine reduces
the fluorescence intensity of riboflavin by complex formation).
iv. COLLISION QUENCHING: -
 It is results of several factors like presence of heavy metals, halides,
increase in temperature and decrease in viscosity where the number
of collisions increased. Hence quenching takes place.
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23. CONCLUSION
 Fluorescence intensity measurement allows the determination of
the presence of fluorophores and their concentrations.
Fluorescence intensity measurement is used in numerous
biochemical assays.
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24. REFERENCES
Dr.S. Ravi Sankar. The text book of pharmaceutical analysis, Fourth
Edition.
Joseph R. Lakowicz. Principles of Fluorescence Spectroscopy, Third
Edition.
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25. Thank you…
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