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RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
Presented by
Ms. K. Sandhya Rani
(Reg.No:20L81S0702)
Under the guidance of
Dr. P.Ramalingam M.Pharm, Ph.D.,
Director – R&D Division
& Professor of Pharmaceutical and Medicinal Chemistry,
President – IPA Local Branch – Anantapuramu.
Quenching in Fluorimetry
A Seminar as a part of curricular requirement for 1st year
M. Pharm 1st semester
RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2
 Introduction
 Quenching
 Types of Quenching
 Applications
 Conclusion
 References
Contents:
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3
Fluorescence:
 When a molecule absorbs light in the visible or ultraviolet range of
the spectrum, it is excited from the electronic ground state to an
excited state.
 It return to the ground state by releasing the absorbed energy in the
form of heat.
 The emitted light is called Fluorescence.
Introduction:
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RIPER
AUTONOMOUS
NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5
Quenching refers to any process that decreases the fluorescence
intensity of a given substance.
 Eg:- pH, concentration, temperature, viscosity, presence of oxygen,
heavy metals or specific chemical substances etc.
Quenching takes place due to presence of an anion or ions with
loosely bound electrons.
During Quenching there is no permanent reaction between the
fluorescent substance and the quenchers.
Quenching:
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 Quenchers are substances capable of absorbing energy from a
fluorophore (such as a fluorescent dye) and re-emitting much of that
energy as either heat (in the case of dark quenchers) or visible light
(in the case of fluorescent quenchers).
Eg: Molecular oxygen, Thiocyanate, Halogens, Disulfide,
Nitric oxide.
Quenchers:
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7
 Fluorophores are fluoroscent chemical compounds that can re-emit
light upon excitation.
It absorbs light energy of specific wavelength and re-emits at longer
wavelength.
Fluorophores:
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1. Concentration Quenching
2. Chemical Quenching
3. Static Quenching
4. Collisional Quenching
Types of Quenching:
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Concentration Quenching may be caused by excessive absorption of
either primary or fluorescent radiation by the solution.
This is also called as Inner Filter effect.
It is a kind of Self Quenching.
 It can be calculated by following equation:
F=QI0act
1.Concentration Quenching:
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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10
In the above equation,
Q = Fluorescence efficiency (Constant for a particular substance)
I0 = Incident light (Constant for a instrument)
a = Molecular extinction coefficient, which is constant for a substance
t = Path length
c = Concentration of the substance
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Deviations at higher concentrations can be
attributed to self-quenching.
Concentration of the Solution
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 Chemical quenching is due to various factors like changes in pH,
Presence of oxygen, halides and electron withdrawing groups, heavy
metals etc.,
PH: Aniline at pH 5 to 13 gives blue fluorescence when excited at
290nm.
But at pH <5 and >13 it does not exhibit fluorescence.
Oxygen: Presence of oxygen leads to quenching because of its
paramagnetic property.
2. Chemical Quenching:
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 Halides and electron withdrawing groups: Halides like chlorine,
bromine, iodide and electron withdrawing groups like nitro and
carboxylic group leads to quenching.
3. Static Quenching:
 It occurs at the ground state of Fluorescing molecule.
 Also called Contact quenching
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 It involves the formation of chemical complex between the
fluorophore and quencher.
 Ex: Caffeine and related xanthines and purines reduce intensity
of riboflavin by static mechanism.
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It occurs by the interaction of a quencher molecule with an
excited molecule of the fluorescing substance.
Also called Dynamic quenching.
 Ex: Halides such as chloride or, iodides are well know collisional
quenchers.
4. Collisional Quenching:
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Determine Membrane permeability to the quenchers.
Determination of diffusion constant.
Investigation of conformational states of proteins.
Used as a indicator of DNA hybridization.
Applications:
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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 17
Quenching is an undesirable effect an the possibility of encountering
this type of interference should be evaluated in developing a
fluorometric assay.
However, this phenomenon can be used as an analytical means for
determining the concentration of the compounds known to quench
fluorescence.
Conclusion:
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 18
1. Douglas A Skoog, Donald M west, F James holler, Stanley R
crouch, Molecular fluorescence spectroscopy, Text book of
Fundamentals of analytical chemistry;8: 826-838.
2. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed.,
Springer, New York;2006: 954.
3. Kautsky H, Quenching of luminescence by oxygen. Trans Faraday
Soc;35: 216–219.
4. Kommu Naresh, Applications of Fluorescence spectroscopy,
journal of chemical and Pharmaceutical sciences;2004: 18-21.
References:
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 19

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Quenching In Fluorimetry.

  • 1. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1 Presented by Ms. K. Sandhya Rani (Reg.No:20L81S0702) Under the guidance of Dr. P.Ramalingam M.Pharm, Ph.D., Director – R&D Division & Professor of Pharmaceutical and Medicinal Chemistry, President – IPA Local Branch – Anantapuramu. Quenching in Fluorimetry A Seminar as a part of curricular requirement for 1st year M. Pharm 1st semester
  • 2. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2  Introduction  Quenching  Types of Quenching  Applications  Conclusion  References Contents:
  • 3. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3 Fluorescence:  When a molecule absorbs light in the visible or ultraviolet range of the spectrum, it is excited from the electronic ground state to an excited state.  It return to the ground state by releasing the absorbed energy in the form of heat.  The emitted light is called Fluorescence. Introduction:
  • 4. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4
  • 5. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5 Quenching refers to any process that decreases the fluorescence intensity of a given substance.  Eg:- pH, concentration, temperature, viscosity, presence of oxygen, heavy metals or specific chemical substances etc. Quenching takes place due to presence of an anion or ions with loosely bound electrons. During Quenching there is no permanent reaction between the fluorescent substance and the quenchers. Quenching:
  • 6. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6  Quenchers are substances capable of absorbing energy from a fluorophore (such as a fluorescent dye) and re-emitting much of that energy as either heat (in the case of dark quenchers) or visible light (in the case of fluorescent quenchers). Eg: Molecular oxygen, Thiocyanate, Halogens, Disulfide, Nitric oxide. Quenchers:
  • 7. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7  Fluorophores are fluoroscent chemical compounds that can re-emit light upon excitation. It absorbs light energy of specific wavelength and re-emits at longer wavelength. Fluorophores:
  • 8. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 8 1. Concentration Quenching 2. Chemical Quenching 3. Static Quenching 4. Collisional Quenching Types of Quenching:
  • 9. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9 Concentration Quenching may be caused by excessive absorption of either primary or fluorescent radiation by the solution. This is also called as Inner Filter effect. It is a kind of Self Quenching.  It can be calculated by following equation: F=QI0act 1.Concentration Quenching:
  • 10. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10 In the above equation, Q = Fluorescence efficiency (Constant for a particular substance) I0 = Incident light (Constant for a instrument) a = Molecular extinction coefficient, which is constant for a substance t = Path length c = Concentration of the substance
  • 11. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11 Deviations at higher concentrations can be attributed to self-quenching. Concentration of the Solution
  • 12. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 12  Chemical quenching is due to various factors like changes in pH, Presence of oxygen, halides and electron withdrawing groups, heavy metals etc., PH: Aniline at pH 5 to 13 gives blue fluorescence when excited at 290nm. But at pH <5 and >13 it does not exhibit fluorescence. Oxygen: Presence of oxygen leads to quenching because of its paramagnetic property. 2. Chemical Quenching:
  • 13. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 13  Halides and electron withdrawing groups: Halides like chlorine, bromine, iodide and electron withdrawing groups like nitro and carboxylic group leads to quenching. 3. Static Quenching:  It occurs at the ground state of Fluorescing molecule.  Also called Contact quenching
  • 14. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 14  It involves the formation of chemical complex between the fluorophore and quencher.  Ex: Caffeine and related xanthines and purines reduce intensity of riboflavin by static mechanism.
  • 15. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 15 It occurs by the interaction of a quencher molecule with an excited molecule of the fluorescing substance. Also called Dynamic quenching.  Ex: Halides such as chloride or, iodides are well know collisional quenchers. 4. Collisional Quenching:
  • 16. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 16 Determine Membrane permeability to the quenchers. Determination of diffusion constant. Investigation of conformational states of proteins. Used as a indicator of DNA hybridization. Applications:
  • 17. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 17 Quenching is an undesirable effect an the possibility of encountering this type of interference should be evaluated in developing a fluorometric assay. However, this phenomenon can be used as an analytical means for determining the concentration of the compounds known to quench fluorescence. Conclusion:
  • 18. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 18 1. Douglas A Skoog, Donald M west, F James holler, Stanley R crouch, Molecular fluorescence spectroscopy, Text book of Fundamentals of analytical chemistry;8: 826-838. 2. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd ed., Springer, New York;2006: 954. 3. Kautsky H, Quenching of luminescence by oxygen. Trans Faraday Soc;35: 216–219. 4. Kommu Naresh, Applications of Fluorescence spectroscopy, journal of chemical and Pharmaceutical sciences;2004: 18-21. References:
  • 19. RIPER AUTONOMOUS NAAC & NBA (UG) SIRO- DSIR Raghavendra Institute of Pharmaceutical Education and Research - Autonomous K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 19