PerkinElmer 200A Series, User Manual

Page: 45 / 172

System Description
45
Figure 3-5 shows the energy level transitions of an organic molecule for the various processes involved in
absorption, fluorescence and phosphorescence.
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.
When a dilute sample is used, the intensity of fluorescence is expressed by:
F = KI
o
c λεϕ
F : Fluorescence intensity
K : Instrumental constant
I
o
: Intensity of exciting radiation
c : Concentration of the compound of interest
λ
: Optical path length of cell
ε : Absorptivity of substance
ϕ
: Quantum efficiency of substance
Advantages of Fluorometry
Fluorescence can provide a significantly greater degree of sensitivity than absorbance measurements.
This increase in sensitivity is due to the fact that in fluorescence, the signal due to the compound of
interest is measured relative to the fluorescence of the blank (which is zero). In contrast, absorbance
measurements compare the transmittance of the solution of the compound of interest relative to the
transmittance of the blank. As the concentration of the solution falls, the transmittance of the sample and
the blank become more similar.