CHM 3402 Experiment 6

Result
Fluorescence spectrum absorption UV-Vis
compound Fluorescence Absorbance λmax / nm

emission spectrum ABS
range
Quinine sulfate 360-600nm 0.195 349.5
Fluorescein 450-650nm 0.336 490.5
Eosin 500-650 0.074 515.5
Rhodamine B 500-650 0.121 553.5

Calculation
Calculation
Spectrum fluorescence
Area of Quinine sulphate = area A + area B

= (1/2 x 6.5 x 3.7) cm2 + (1/2 x 6.5 x 4.3) cm2
= 12.02 +13.975
=25.99cm2
Area of Fluorescene = area C + area D

= (1/2 x 9.8x 1.8 ) cm2 + (1/2 x 9.8 x 2.5 ) cm2
= 8.82 cm2 +12.25cm2
= 21.07 cm2
Area of Rhodamine B = area E + area F

= (1/2 x 2.4 x 11.3 cm2) + (1/2 x 2.5x 11.3 ) cm2
= 13.56 cm2 +14.13 cm2
=27.69 cm2
Area of Eosin = area G + area H

= (1/2 x 8.25 x 1.4 ) cm2 + (1/2 x 8.25x 2.3 ) cm2
= 5.775 cm2 +9.49 cm2
=15.26 cm2
Calculation of Quantum yield
Quantum yield of Fluorescein

Фfu = Ffu x AQ x ФfQ
Ffq x Au
= 21.07 x 0.195 x 0.54

25.99 0.336
= 0.2540
Quantum yield of Rhodamine B

Ffq x Au
= 27.69x 0.195 x 0.54

25.99 0.121
= 0.9268
Quantum yield of Eosin

Ffq x Au
= 15.26 x 0.195x 0.54

25.99 0.074
= 0.8430
Discussion
Spectrum fluorescence of each compound will let us to know the area of the graph.All
of the compounds spectrum were obtained and the area of compounds were calculated to get
the quantum yield. The skill of using UV-Vis spectrophotometer and fluorescene
spectrophotometer was learned. We found that from this quantum yield experiment was the
deviation of quantities molecules was as same as the quantities of excited molecules.
Actually the precise of the absorbance spectrum was affected by several factors. They
were: temperature changing, pH, dilution of oxygen, structural and others. But the absorbance
was not affected by concentration mixture.The dilution was done properly to make sure that the
absorption at λmax was precise and not more than 0.05abs.
If the solution was not prepared well then the error of the wavelength will be got and it
will affect the calculation and the value quantum yield was not precise too. That is why all of
the test tube and apparatus like bikers and tube for the UV-Vis and fluorescene spectrometer
must be clean and dry always.
When a fluorophore absorbs a photon of light, an energetically excited state is formed. The
fate of this species is varied, depend on the nature of the fluorophore and its surroundings, but
the end result is deactivation (loss of energy) and return to the ground state.
The fluorescence quantum yield is the ratio of photons absorbed to photons emitted through
fluorescence. In other words the quantum yield gives the probability of the excited state being
deactivated by fluorescence rather than by another, non-radiative mechanism.
The measurement of fluorescence quantum yields can often be difficult.
The absorption of UV or visible radiation corresponds to the excitation of outer electrons.

There are three types of electronic transition which can be considered;
1. Transitions involving π , σ , and n electrons

2. Transitions involving charge-transfer electrons
3. Transitions involving d and f electrons (not covered in this Unit)
When an atom or molecule absorbs energy, electrons are promoted from their ground state to
an excited state. In a molecule, the atoms can rotate and vibrate with respect to each other.
These vibrations and rotations also have discrete energy levels, which can be considered as
being packed on top of each electronic level.
Absorbing species containing π , σ , and n electrons
Absorption of ultraviolet and visible radiation in organic molecules is restricted to certain

functional groups (chromophores) that contain valence electrons of low excitation energy. The
spectrum of a molecule containing these chromophores is complex. This is because the
superposition of rotational and vibrational transitions on the electronic transitions gives a
combination of overlapping lines. This appears as a continuous absorption band
Possible electronic transitions of π , σ , and n electrons are;
Precaution steps:
1. the dilution must be done properly .
2. all of the apparatus must be clean and dried well.
3. the mixture of compounds and solvent must be stired
4. Fluorescence emission spectrum range must be changed during testing the different
compound.
5. the error of solvent must be made sure whether had or not during the UV-Vis
spectrophotometer was used.
Conclusion
1. Φfu of fluoresin was
2. Φfu for eosin was
3. Φfu for Rhodamine B was
Φfu for quinine sulphate was
Reference
a) Silverstein, R. M, G.C. Basster and T.C. Morrill (1981)
Spectrometric Identification of Organic Compounds
4th Edition New York : John Wiley & Sons
b) John A. Landgrebe, 1993

Theory and Practice in the Organic Laboratory
with Microscale and Standard Scale Experiments
4th Edition Pacific Grove, California
c) Instrumental Method of Analysis- Willard H.H, L.L.Marntt

CHM 3402 Experiment 6

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Result

Fluorescence spectrum absorption UV-Vis

compound Fluorescence Absorbance λmax / nm

Fluorescein 450-650nm 0.336 490.5

Eosin 500-650 0.074 515.5

Rhodamine B 500-650 0.121 553.5

Area of Quinine sulphate = area A + area B

Area of Fluorescene = area C + area D

Area of Rhodamine B = area E + area F

Area of Eosin = area G + area H

Quantum yield of Fluorescein

= 21.07 x 0.195 x 0.54

Quantum yield of Rhodamine B

= 27.69x 0.195 x 0.54

Quantum yield of Eosin

= 15.26 x 0.195x 0.54

The measurement of fluorescence quantum yields can often be difficult.

The absorption of UV or visible radiation corresponds to the excitation of outer electrons.

1. Transitions involving π , σ , and n electrons

Absorbing species containing π , σ , and n electrons

Absorption of ultraviolet and visible radiation in organic molecules is restricted to certain

b) John A. Landgrebe, 1993

c) Instrumental Method of Analysis- Willard H.H, L.L.Marntt

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