The document discusses fluorescence and its applications in drug analysis, including techniques like fluorometry and the quenching process that reduces fluorescence intensity due to various factors such as pH, temperature, and the presence of chemicals. It details types of quenching, including collisional, static, and chemical quenching, and explains how these phenomena can impact fluorescence measurements. Lastly, it emphasizes the use of quenching as both a challenge in assay development and a potential analytical tool for determining concentrations of compounds.

1. Fatema Tuz Zohora
22nd batch,
Department of Pharmacy,
University of Science and Technology Chittagong (USTC).

2. Fluorescence
 Emission of visible light by a
substance after absorbing any
external radiation is known as
fluorescence.
 There are many drugs and other
chemical compounds that show
fluorescence property. Such as,
riboflavin, vitamin-A, aniline,
quinine, fluorescein etc.
Fig: Saturated solution of 0.025g riboflavin
in 200 ml water after UV exposure.

3. Fluorometry
 The intensity and composition of emitted radiation can be measured by
a sensitive method of analysis which is called fluorometry.
 This method is significant in :
 Analysis of many drug substances like riboflavin, thiamine,
reserpine, quinine etc.
 Analysis of trace amount of drugs, metabolites, proteins,
hormones, steroids etc. in tissues and fluids.
 Fluorometry is also being used in the treatment and diagnosis
of cancer in recent years.

4. Quenching of fluorescence
 Quenching refers to any
process that reduces the
fluorescence intensity of a
given substance.
 This may occur due to
various factors like pH,
concentration, temperature,
viscosity, presence of
oxygen, heavy metals or,
specific chemical
substances etc.
Fig: Quenching of quinine fluorescence
in presence of chloride ions

5. Example of quenching agents
Quenching agents Typical fluorophores
Thiocyanate Anthracene
Chloride Quinine
Iodide Tryptophan
Disulfide Tyrosine
Nitric oxide Naphthalene

6. Fig: Types of quenching process
Quenching
Collisional quenching
Static quenching
Concentration quenching
Chemical quenching

7. Collisional quenching
Collisional quenching occurs by the interaction of a quencher molecule
(Q) with an excited molecule of the fluorescing substance (F*).
A simplified mechanism can be written to describe this process:
F + hv F*
F* F + hv
F* + Q F + Q*
Here, the interaction results in the dissipation of excitation energy by a
non radiative energy transfer from F* to Q without or, less fluorescence.

8. Fig : Simple mechanism of collisional quenching
Halides ions such as chlorides or, iodides are well known collisional
quenchers. For example, quenching of quinine drug by chloride ion
or, quenching of tryptophan by iodide ion follow collisional
quenching process.
Weak coupling
Light
Energy transfer
Quenching of light
F* Q
Distance

9. Static quenching
Static quenching occurs at the ground state of fluorescing molecule.
It can be simplified by following mechanism:
F + Q F : Q
F:Q + hv Q* + F
Q* Q + energy
Here, a complex formation occurs between the fluorescing molecule at
the ground state (F) and the quencher molecule (Q) through a strong
coupling. Such complex may not undergo excitation or, may be excited to
a little extent reducing the fluorescence intensity of the molecule.

10. Strong coupling
Fig: Static quenching
Caffeine and related xanthines and purines reduce intensity of
riboflavin by static mechanism. Quenching that occurs due to oxygen
also follows this mechanism.
Light Energy transfer
F Q
Quenching
of light

11. Concentration quenching
Concentration quenching is a
kind of self quenching.
It occurs when the
concentration of the
fluorescing molecule increases
in a sample solution. The
fluorescence intensity is
reduced in highly concentrated
solution ( >50 μg/ml ).
100 200 300
concentration (µg/ml)
Fig: Influence of concentration on the
fluorescence of phenol solutions.
80
60
40
20
0
fluorescenceintensity

12. Chemical quenching
Chemical quenching is due to various factors like change in pH,
presence of oxygen, halides and electron withdrawing groups, heavy
metals etc.
 Change in pH : Aniline at pH (5-13) gives fluorescence
when excited at 290 nm. But pH <5 or, pH >13 it does not
show any fluorescence.
 Oxygen : Oxygen leads to the oxidation of fluorescent
substance to non fluorescent substance and thus, causes
quenching.

13.  Halides and electron withdrawing groups : Halides like
chloride ions, iodide ions and electron withdrawing groups like
-NO , -COOH , -CHO groups lead to quenching.
 Heavy metals : presence of heavy metals also lead to
quenching because of collision and complex formation.
2
Fig: electron withdrawal process by
nitro group

14. Conclusion
In the usual case, quenching is an undesirable effect and the possibility
of encountering this type of interference should always 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. Quenching study can also be used to reveal the
localization of fluorophores in proteins or, membranes and their
permeability to the quenchers.

15. References
 Leslie G. Chatten , Pharmaceutical Chemistry Theory and Application
(volume-2), CBS publishers and distributors, 2014 revised edition, page:
180,181,182,183.
 Joseph R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd edition
(2006), Springer Science publication, page: 278,282,283.
 Wikipedia//Quenching(Fluorescence).

16. Thank you…