In this slide contains types of Quenching and its applications. Presented by: K. Sandhya Rani (Department of pharmaceutical analysis). RIPER,anantpur.

1. 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 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:

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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:

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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:

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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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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:

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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:

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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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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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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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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 19