Hitachi CHROMASTER 5160, Instruction Manual

Page: 447 / 1022

When light strikes an organic molecule in the ground state, it
absorbs radiation of specific wavelengths and several excited
states are populated. A part of the excitation (absorbed) energy
is lost in vibrational relaxation, i.e. radiationless transition to the
lowest vibrational level in the excited state.
The molecule can return to the ground state by;
(1) Emitting radiation (Fluorescence)
(2) Undergoing a radiationless transition to populate the triplet
state. The triplet state can emit radiation
(Phosphorescence). Generally phosphorescence persists
for 10
-4
seconds or longer due to the selection rule imposed
on the triplet-to-singlet transition. In contrast, fluorescence
takes place over a period of 10
-8
to 10
-9
seconds.
(3) Going through a radiationless transition to return directly to
the ground state.
Since a part of the radiation absorbed by the substance is lost as
vibrational energy; the energy emitted by the excited state is less
than that absorbed by the compound (i.e. the fluorescence
wavelength is longer than the excitation wavelength,
*Stokes’
Law
).
The ratio of the number of photons emitted during fluorescence
to the number of photons absorbed is called the quantum
efficiency of fluorescence (Fluorescence Yield). If two
compounds absorb the same number of photons, the
fluorescence intensity of the compound with the larger
fluorescence quantum yield will be greater than that from a
compound with a lower fluorescence quantum yield. Also, the
intensity of the fluorescence emitted by a compound is
proportional to the number of photons absorbed by it.
Therefore, when a dilute sample is used, the intensity of
fluorescence is expressed by:
F = KI
o
c
l
εϕ
F : Fluorescence intensity
K : Instrumental constant
I
o
: Intensity of exciting radiation
c : Concentration of the compound of interest
l : Optical path length of cell
ε
: Absorptivity of substance
ϕ
: Quantum efficiency of substance
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