Molecular

Fluorescence
and
Phosphorescence
Principle
 Process
 Molecular absorption of photons in the UV–Vis
range causes the electronic transition from a lower
energy level [typically the lowest vibrational level v0, of
the ground electronic singlet state (S0)] to an excited
singlet state S1.

 The molecule rapidly undergoes vibrational

relaxation to the lowest vibrational level of the excited
state from which it may be de-excited by one of several
competitive pathways. De-excitation directly back to the
ground state can occur either by emission of photons.:

Fluorescence.
 Alternatively, under favorable
conditions, the molecule may undergo a “forbidden”
vibrational relaxation to the lowest vibrational level of To or
return to S1 by intersystem crossing. In the latter case, de-
excitation by photon emission results in delayed
fluorescence, which is spectrally identical to prompt
fluorescence, but it exhibits the longer decay associated
with

Phosphorescence
Instrumentation
1. Light Sources

 The xenon arc lamp

 High pressure mercury arc lamp
 Halogen lamps and combination xenon–
mercury lamps
 Laser
2. Wavelength Selectors

 Either filters or monochromators can be

used for wavelength selection
 A filter is used in the excitation beam
along with a monochromator in the
emission beam to collect emission
spectra.
3. Sample Compartments

 Cuvettes for fluorescence measurements

of solutions are usually rectangular with at
least two adjacent faces that are
transparent.
 Disposable acrylic cuvettes can be used
for the 275–350 nm wavelength
 Cuvettes are available for microvolume
work
4. Detectors

 Photomultipliertubes(PMTs)
 In the range of 200–600 nm, with
maximum sensitivity 300-500 nm obtained
in also available for detection above 600
nm.
 Cooled to temperatures as low as –40 C
to minimize temperature-dependent
noise.
Phosphorimeter
 Phosphorescence can be measured with
a conventional, continuous source
fluorescence instrument
 Equipped with a phosphoroscope
attachment.
 The phosphoroscope is used to eliminate
fluorescence signals on the basis of the
large difference in lifetimes between
fluorescence and phosphorescence
Sample
 Solution
 exhibitnative luminescence include
organic molecules with conjugated bond
systems, such as polycyclic and
polynuclear aromatic hydrocarbons
 fluorescent metal chelates,
 certain rare earth elements, and
inorganic lanthanide and
 uranyl compounds. S
Application
 Chemical Analysis and Characterization
 Luminescence Detection in Chemical
Separations
 Biological Applications
Calculation