BIKRAM MONDAL

ROLL NO-202122013

Objective : Calculation of Stern-Volmer

constant and Determination of Analyte
concentration using Steady state
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. From there it can
return to ground state by releasing the absorbed energy in the
form of heat and by radiation in the visible or near-infrared
spectral range. The emitted light is called fluorescence .
Fluorescence can be detected with very high sensitivity even from
single molecules and it is used in a large number of chemical and
biochemical applications. Sensitive fluorescence detection relies on
the fact that the emitted light usually has a longer wavelength
than the intense light used for excitation. This_difference between
absorption and fluorescence wavelength (maxima) is also known
as shift and can be understood in the following way: in addition
to the change of electronic structure absorption can also lead to

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the excitation of vibrational levels, which requires more energy or
light of shorter wavelength.

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Flouroscence quenching can be divided into two ways

Static quenching: this is due to the formation of a non flurescent

complex between fluorophore and quencher at the ground state.

Dynamic quenching: an excited state fluorophore interact with quencher

and as a result in a non radiative deactivation known as dynamic
quenching.the efficiency of this process is dependent on concentration
of quenching species.

This type of quenching occurs via two ways

i)forster mechanism ii)dexter mechanism

: Charge fluctuations in donor and acceptor can

influence each other over distances of the order of 10 nm if they occur near
resonance of an electronic transition in both molecules (transition dipole
interaction). The probability of energytransfer in this case is proportional to

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the overlap between the emission spectrum of thedonor and the absorption
spectrum of the acceptor and decreases with donor-acceptordistance R like
1=R6. This mechanism is responsible for the transfer of energy from the
light-collecting antenna complexes to the reaction centre in natural
photosynthesis.

Dexter Mechanism : When donor and acceptor come sufficiently close

for their orbitals to overlap, the excited electron of the donor can be
transferred to an unoccupied
orbital of the acceptor. At the same time, an electron of the acceptor moves
to the HOMO of the donor. This process is only effective for donor-acceptor
distances smaller than 15_A. A common variant of this process is triplet
quenching, when the donor is initially in the T1 state.] During reductive
quenching the quencher transfers an electron to the excited molecule which
stops to fluorescence.

Fluorescence is example of intramolecular deactivation (quenching)

processes. An intermolecular deactivation is where the presence of
another chemical species can accelerate the decay rate of a chemical in
its excited state. In general, this process can be represented by a simple
equation

� ������� + � → � + �
A is chemical species,Q is quencher
The kinetics of this process follows the Stern volmer relationship
��°
= 1 + ���° �
�
Io/I=1+Ks �

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Io=fluoroscent intensity without quencher; I=Fluoroscent intensity with quencher
Ks=stern volmer constant; � =Concentration of quencher
The stern volmer equation allows us to determine the product ���° = ��
from the slope of a plot of ��/� against the quencher concentration �

APPLICATION OF FLUORESCENCE: 1) The three dimensional structure of protein

can be studied by measuring the proximity of known fluolorescent groups within
the protein.
2)Study interaction between biomolecules.
3)Determination of analyte concentration.

OBSERVATION:when we start adding the quencher to the flurorophore

sample the intensity as expected start to decrease from the previous
values.more the concentration of quencher lesser is the value of
intensity.we observe a maximum intensity at a certain wavelength.