Jenway 6285 - Manual (Page 12)

Section 3
Fluorescence Theory
The interaction between electromagnetic radiation and matter provides a useful,
qualitative and quantitative analytical tool, known as spectroscopy. The region of the
electromagnetic spectrum, to which matter under investigation is subjected to,
defines the type of transitions that occur within the molecules.
Fluorimetry uses radiation from the UV-Vis region of the electromagnetic spectrum to
study transitions between electronic levels in a molecule or atom. The absorption of
energy from light radiation (photons) by a molecule or atom, promotes electrons from
a low energy ground state to a higher energy excited level. This is known as
excitation and the amount of energy transferred to the molecule or atom will depend
on two main factors. The composition of the matter under investigation and the
energy and wavelength of the radiation, have a significant effect on the transitions of
electrons.
The molecule or atom converts the excitation energy to vibrational or light energy and
the electron returns to its ground state. Vibrational energy is transferred through
movement and collision with other molecules, but energy not lost in this way is
released as light radiation. The light emission is known as fluorescence and if some
energy has been removed through vibration, it will be of a lower energy and longer
wavelength than the excitation energy. The wavelength and intensity of the emitted
radiation is dependent on the structure and composition of the molecule and the
excitation wavelength used.
Relationship between concentration and fluorescence
The fluorescence signal F and concentration C of the matter under investigation, are
related by:
F = KQP
0
(1-10 -
ε
bC )
Where
K = A constant characteristic of the instrument (Including instrument
electronics, pH and Temperature)
Q = Quantum efficiency (= Photons emitted/Photons absorbed)
P
0
= Power of incident radiation
ε
= Molar absorptivity of the species (matter)
b = Absorption path length
If the concentration of the matter in question is low (dilute),
ε
bC is small. The
relationship is then linear and the equation can be written as
F = 2.3KQP
0
ε
bC
The accuracy of fluorescent measurements is very high because the radiant energy
being formed is measured directly. There are also only a few, easily controlled limits
on the sensitivity of fluorescence measurements. From the equation above it can be
seen that adjustments made to instrument electrical noise and competing radiations,
along with physical limitations such as radiation energy, sample volume and cell size
affect the measurement sensitivity.
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