MOLECULAR FLUORESCENCE
SPECTROSCOPY
• Fluorescence is a form of photoluminescence; and this
later is a type of luminescence that occurs when certain
molecules are excited by electromagnetic radiation and
as a consequence remission of radiation either of the
same wavelength or longer one takes place.
• The two most common photoluminescence are
fluorescence and phosphorescence which are produced
by different mechanisms.
• Fluorescence is distinguished from phosphorescence by
the lifetime of the excited state, with fluorescence the
excited state ceases immediately after irradiation is
discontinued, (10-7 s), while phosphorescence continued
for a detectable time (100 s).
Theory of molecular fluorescence
• An excited molecule can return to its ground
state by combination of several mechanistic
steps. Deactivation or relaxation processes can
be classified to radiative and nonradiative
processes.
Radiationless deactivation;
1-Vibrational relaxation (VR)
Conversion of the excited electron from the highest energy sublevel to
the lowest energy sublevel in the same main energy level.
2-Internal conversion (IC):
It is intermolecular processes by which a molecular passes from an
electronic excited energy level (S2) to another lower excited energy
level (S1).
3-External conversion (EC):
It is deactivation of an excited electronic state which involve
interaction and energy transfer between the excited molecules and the
solvent or other solutes.
• Intersystem crossing (ISC) is a process in which
the spin of an excited electron is reversed. The
probability of this transition is enhanced if the
lowest vibrational energy level of the lowest
excited singlet state is almost identical in its
energy to that of the triplet excited state.
• ISC is common in molecules containing heavy
atoms such as iodine and bromine, also it is
enhanced in presence of paramagnetic molecules
such as molecular oxygen.
Radiative deactivation:
1-Fluorescence
Transition from S2 or S1 to the ground singlet state (S0)
occurs with loss of energy in the form of EMR (emission
of photons) is termed fluorescence (S1 or S2-S0).
2-Phosphorescence
Phosphorescence occurs when an electron in an
excited triplet state relaxes to the ground singlet state
while emitting radiation (T1 – S0).
Excitation and emission spectra:
If the intensity of emitted light (fluorescence) at a
fixed wavelength (emission) is plotted as a function
of wavelength of radiation used to excite a
molecule, an excitation spectrum will result.
On the other hand, if the intensity of emitted
radiation (fluorescence) is plotted versus
wavelength, an emission spectrum is obtained. In
this case, the sample is irradiated with
monochromatic radiation of certain wavelength
(excitation) and a scan of the wavelength of emitted
radiation is recorded.
If both of the excitation and emission spectra of a compound are
plotted on the same chart, the following will be observed: 1displacement of emission band to longer wavelength (Stock’s
shift). 2- excitation and emission spectra bear a mirror image
relationship to each other as shown in the following figure.
Excitation and emission spectra
Quantum yield ():
The quantum yield or quantum efficiency () for a fluorescent
process is the ratio of the number of molecules that fluoresce to
the total number of excited molecules or the ratio of number of
photons emitted to that absorbed. For a highly fluorescent
molecule  approachs unity ( = 1), while for a nonfluorescent
molecule =0.
Quantitative fluorimetry:
F = 2.3 K bc I0
F = K/ c
A plot of fluorescence intensity versus concentration is linear at
low concentration. When the concentration becomes high
enough, A > 0.05 linearity is lost.
At high concentration, two main factors are responsible
for deviation from linearity:
1-Self-absorption: this occurs when the wavelength of
emission overlaps with an absorption peak. Then, some
of the emitted radiation will be absorbed by molecules in
solution and a decrease in fluorescence takes place.
2- Self-quenching: it results from the collision of the
excited molecules.
Factors affecting fluorescence:
1- Molecular structure
• The most intense and most useful fluorescent behavior is
found in compounds containing aromatic functional
group. Compounds containing aliphatic and alicyclic
carbonyl groups or conjugated double-bond structures
may also exhibit fluorescence.
• The quantum yield increases with the increase of number
of fused rings. The simplest heterocyclics, such as
pyridine, thiophene, pyrrole and furan do not fluoresce
(the lowest transition is n - * system which is rapidly
converted to triplet and prevents fluorescence).
• Halogen substitution especially with bromine and iodine
results in a decrease in fluorescence due to intersystem
crossing.
• Fluorescence is favored in molecules that posses rigid planer
structure. For example fluorene fluoresce much more intense
than biphenyl due to rigidity furnished by methylene group in
fluorene. The influence of rigidity is accounted for the
increase of fluorescence of certain chelating agents when
they form complexes with a metal ion e.g. the fluorescent
intensity of 8-hydroxyquinoline is much increased when it
forms zinc complex.
O
Zn
N
C
H2
Fluorene
n2
Biphenyl
The zinc complex
2- Effect of temperature and solvent:
• The quantum efficiency of fluorescence by most molecules
decreases with increasing temperature, as deactivation by
external conversion is favored. Also a decrease in solvent
viscosity leads to the same result.
• Polar solvents may enhance fluorescence, while it is
decreased by solvents containing heavy atoms such as carbon
tetrabromide or ethyl iodide.
3- Effect of dissolved oxygen:
Being paramagnetic, dissolved oxygen decreases the
fluorescence due to intersystem crossing.
Instrumentation
It is composed of the following main parts shown in
the following diagram
Schematic diagram of a spectrofluorimeter
1-Source of energy
Several sources have been used, the two most
commonly used are:
A-Mercury–arc lamp:
It is a quartz lamp containing mercury vapor which
upon electrical excitation emits line spectra of
several definite wavelengths. It can not be used
when a scan of spectrum is required.
B-High pressure xenon lamp:
This lamp emits a continuum of radiation
throughout the UV-Vis region so it is useful when
spectrum scanning is required.
2-Wavelength selector
Two filters (either absorption or interference filters can be
used) or monochromators (grating type) are used; one
between the source and the sample and the other between
the sample and the detector.
3-The cell:
Tetragonal or cylindrical transparent, glass or quartz tubes
are used (the four sides are transparent).
Compare with the sample cell in the spectrophotometry.
4-Detectors and readout meter:
Photomultiplier type is used since the intensity of emitted
radiation is small. Digital or analog or null point meter are
used.
Important notes:
• Emission of radiation by sample takes place in all
directions. The emitted radiation is measured at 900
from the path of the exciting beam and at the center of
the cell.
• This is to minimize the error due to scattering of light
from the walls of the cell and to prevent the
interference from the exciting beam, which occurs at
other angles.
• Since a broad emission band is obtained, it is
necessary to use a second wavelength selector
between the sample and the detector in order to pass
the most intense emitted wavelength (emission).
Applications of fluorimetry:
• Compounds which are intrinsically fluorescent are
easily determined at very low concentrations by simple
fluorimetric method (Direct fluorimetry). For example,
phenobarbitone, quinine, emetine, adrenaline,
cinchonine, reserpine vitamin A, riboflavine and other
natural products.
• Nonfluorescent substances can be determined after
chemical reaction (Indirect fluorimetry).
• Inorganic ions can be determined either by formation
of fluorescent chelates upon reaction with fluorimetric
reagents e.g. 8- hydroxyquinoline (for Al3+), benzoin
(for Zn2+) or flavanol (for Zr3+) or by measuring the
quenching of fluorescence of a fluorescent substance
in the presence of some ions.