Experiment 8: Determination of Fluorescence Quantum Yield of an Unknown Fluorophore

Using a Standard Fluorophore (Coumarin 102)

Theory: The fluorescence quantum yield of a compound is defined as the fraction of the
molecules that emit photons after direct excitation by the source. This quantity is not the same as
the total number of emitted photons which escape a bulk sample divided by the total number of
absorbed photons, although in many instances the two quantities are nearly equal. Practically,
absolute quantum yield is important. They allow one to access the sensitivity of a proposed
fluorimetric determination of materials and the extent of interferences.
They are necessary for calculating thresholds for laser action and for judging the suitability of
materials as wavelength shifts in optical pumping experiments or for the use of energy donors.
Yields coupled with fluorescence data are also used for the evaluation of purity of materials.
There are many methods of measuring quantum yields, such as MgO as a standard, solution
scatterer as standards, comparison with compounds of known quantum yields (optically dense
and optically dilute measurements), calorimetric methods etc.
The determination of quantum yield using optically dilute solution is the most common method
for the determination of quantum yield (QY) of an unknown compound.
The Quantum yield were calculated using the following equation.
2

= ( ) ( 2 )


Where, is the quantum yield (QY)
I is the fluorescence area under the curve
n is refractive index of the medium
OD is optical density at the exciting wavelength
S and r represent the parameters for the sample and reference, respectively

Glassware: Volumetric flask ( 100 mL x 2), graduated pipette (10 mL x 1, 2 mL x 1), beaker
(100 mL x 2), glass funnel
Chemicals: Coumarin 102 solution (~10-5 M), Unknown dye(~10-6M) , spectroscopic grade
EtOH
Procedure:

(i) Check the absorbance of supplied Coumarin 102 and note down the value at max. Dilute the
solution using spectroscopic grade Ethanol such that the absorbance of the solution is close to
0.1.
(ii) Record the emission spectra and note down the value of the integrated area under the curve.
(iii) Now check the absorbance of the unknown dye and dilute it using spectroscopic grade
Ethanol such that the absorbance of the solution at max is close to 0.1
(iv) Record the emission spectra of the dye and note down the value of the integrated area under
the curve.
(v) Now determine the quantum yield of the unknown dye using the standard equation.
Quantum yield of Coumarin 102 in EtOH is 0.764
Instrumentation:
excitation = 390 nm
Scan range: 400 nm to 770 nm
Slit: 2/2
Emission correction: S1C/R1
Increment: 1 nm
Integration time: 0.1 s
Note down all the observations in your lab notebook. Clean all your glassware and hand over to
the lab attendant.