FLUORESCENCE SPECTROSCOPY
MOLECULAR FLUORESCENCE
SPECTROSCOPY
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FLUORESCENCE SPECTROSCOPY
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FLUORESCENCE SPECTROSCOPY
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
mechanisms.
which
are
Fluorescence
produced
is
by
different
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). Both monoatomic
particles
and
polyatomic
molecules
fluorescence, in gaseous, liquid and solid
can
undergo
states. When
the radiation absorbed is exactly the same emitted, the
fluorescence
is
known
by
resonance
fluorescence.
Polyatomic molecules or ions exhibit resonance radiation,
in addition characteristic ones of longer wavelength are
emitted this phenomenon is known by Stock’s shift.
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. Figure 1 shows the partial energy
diagram for a photoluminscent
system. The straight
vertical arrows represent the radiative relaxation processes
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FLUORESCENCE SPECTROSCOPY
namely fluorescence and phosphorescence, where release
of photons occurs. The wavy arrows are radiationless
processes. The favored route to the ground state is one
that minimizes the life time of the excited state.
Partial energy diagram for a photoluminescent system.
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FLUORESCENCE SPECTROSCOPY
Radiationless deactivation;
1-Vibrational relaxation:
A molecule may be promoted to any of several
vibrational levels during the electronic excitation. In
solution, the excess vibrational energy is immediately lost
as a consequence
of collision between the excited
molecules and those of the solvent, the result is minute
increase in solvent temperature. This relaxation process is
very rapid (10-12 s).
Accordingly, fluorescence from solutions when occurs
always involves a transition from the lowest vibrational
level to any vibrational levels of the ground state result in
band emission. A consequence of the efficiency of
vibrational relaxation is that the fluorescence band
(emission spectrum) is displaced at longer wavelength
(lower energy) than the absorption band (excitation
spectrum)
2-Internal conversion:
It is intermolecular processes by which a molecular
passes from an electronic excited energy level (S2) to
another lower excited energy level (S1). This occurs when
the lowest vibrational energy level of S2 coincide with one
of the vibrational levels of S1.
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FLUORESCENCE SPECTROSCOPY
3-External conversion :
It is deactivation of an excited electronic state which
involve interaction and energy transfer between the
excited molecules and the solvent or other solutes.
4-Intersystem crossing:
The outer most electrons
in molecular orbitals are
usually even number, paired electrons with no net electron
spin (diamagnetic) This molecular electronic state in which
all electron spins are paired is called a singlet state (in
ground state it is denoted by SO). When one of the
electrons of a molecule is excited to a higher energy level
a singlet or a triplet state can result. In the excited singlet
state (S1 or S2) the spin of the promoted electron is paired
with that in ground state; while still in the excited the
electron may reverse its spin (flipping) the two electrons
become unpaired and thus have the same spinning, these
states can be
represented as follows:
SO
Ground singlet
S1
Excited singlet
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T1
Excited triplet
FLUORESCENCE SPECTROSCOPY
A molecule with an unpaired electron in an excited
level that has a spin that is identical to that of electron in
the ground state, is in the triplet state. Intersystem
crossing is a process in which the spin of an excited
electron is reversed. The probability of this transition is
enhanced if the lowest vibrational of the lowest excited
singlet state are almost identical to the triplet excited
state.
The potential energy of the triplet state is less than
that of the singlet. Intersystem crossing 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:
As mentioned previously, an excited electron in
singlet state looses vibrational energy to reach the lowest
vibrational level by collision, transition from this to the
ground singlet state with lose of energy in the form of
photons i.e. emission of EMR; is known by fluorescence
(S1or S2-S0). Also, sometimes fluorescence occurs
When the electron found in an excited singlet level
(S2) converted to excited singlet state but of lower energy
(S1) by internal conversion, then relaxes to the ground
state with emission of radiation (S1-S0)
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FLUORESCENCE SPECTROSCOPY
2-Phosphorescence:
An electron in the excited singlet state can be
converted to the excited triplet state by intersystem
crossing. Phosphorescence occurs when an electron in an
excited triplet state relaxes to the ground singlet state
while emitting radiation (T1 –S0). The considerable barrier
to the spin reversal that exists in the molecule prevents
intersystem crossing from occurring rapidly as singlet singlet transition. Because of that barrier phosphorescence
occurs on a much longer time scale than fluorescence.
Excitation and Emission Spectra:
λ If the intensity of the fluorescence at one fixed
wavelength (emission) λλis
wavelength
of
the
plotted as a function of
radiation
used
to
excite
the
fluorescence, an activation or excitation spectrum will
result. This will be identical with the absorption spectrum
when corrected for the instrumental effects namely
variation of source out put and
variation of detector
sensitivity to wavelength.
If we plot the intensity of fluorescence obtained when
the sample is irradiated with monochromatic radiation
(excitation) versus wavelength an emission spectrum is
obtained.
If we plot the excitation and emission spectra of a
compound on the same chart the displacement of emission
band to longer wavelength is apparent (Stock’s shift) and
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FLUORESCENCE SPECTROSCOPY
the two spectra bear a ‘mirror image’ relationship to each
other as shown in the following figure.
Excitation and fluorescence spectra of anthracene
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 highly fluorescent molecules it may approach one for
nonfluorescent substance it approaches zero.
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FLUORESCENCE SPECTROSCOPY
Quantitative Fluorimetry and Effect of
Concentration on Fluorescence:
The
power
of
the
fluorescent
radiation
F
is
proportional to the radiant power of the excitation beam
that is absorbed by the system. That is
F= K (I0 -I) K depends upon the quantum efficiency
of the fluorescence process.
To relate F with the
concentration
c of the fluorescence particle
we write
Beer’s law in the form:
 is the molar absorptivity of the
fluorescent molecules  bc is the absorbance A, by
substitution in the first equation F= K I0 (1- 10- bc)
provided  bc = A < 0.05 the exponential term of the
I / I0 =
10-
bc
equation will be :
F = 2.3 K  bc I0 i.e.
F = K/ c. Then a plot of
fluorescent power of a solution versus concentration of the
emitting species should be linear at low concentration c.
When the concentration becomes great enough so that the
absorbance is larger than 0.05, linearity is lost. Two other
factors responsible for further departure from linearity at
high concentration these are:
One-
Self-
absorption;
this
occurs
when
the
wavelength of emission overlaps an absorption peak
then some of the emitted radiation will be absorbed
by the molecules in solution and decrease in
fluorescence takes place.
Two-
Self-quenching; it results from the collision of
the excited molecules.
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FLUORESCENCE SPECTROSCOPY
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.
Most unsubstituted aromatic hydrocarbons fluoresce
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), fusion of
benzene ring in hetero atom results in fluorescent
compound. Halogen substitution specially with bromine
and iodine results in decrease of fluorescence due to
intersystem crossing.
Substitution of carboxylic acid or carbonyl group on an
aromatic ring inhibits fluorescence.
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 8hydroxyquinoline is much increased when it forms zinc
complex.
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FLUORESCENCE SPECTROSCOPY
O
Zn
N
C
H2
Fluorene
n2
Biphenyl
The zinc complex
2- Temperature and Solvent Effects:
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 solvent may enhance fluorescence, while it is
decreased by solvents containing heavy atoms such as
carbon tetrabromide or ethyl iodide.
3- Effect of pH :
The fluorescence of an aromatic compound with
acidic or basic ring substitution is pH dependent. Both
the emission intensity and wavelength
of the ionized
form and the unionized will be different.
4- Effect of Dissolved Oxygen:
Being paramagnetic dissolved oxygen, decrease the
fluorescence due to intersystem crossing.
Instrumentation:
A block diagram of the major components of single
beam
spectrofluorimeter
58
is
shown
in
figure
3.
FLUORESCENCE SPECTROSCOPY
Electromagnetic radiation from an ultraviolet - visible
source passes through a wavelength selector and
through the cell (if monochromators are used the
instrument is called spectrofluorimeter and if filters are
used it is called fluorimeter). Emission of radiation by
sample takes place in all directions.
radiation
The emitted
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 solution, which occurs at other
angles, and to prevent the interference from the exciting
beam.
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).
1-Source of energy:
The source of radiation must be highly intense to
allow considerable excitation. 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 wavelength, it can not be used
when scan of spectrum is required.
b- High pressure Xenon
lamp emits a continuum of
radiation throughout the ultraviolet -visible regions, it is
useful when spectrum scanning is needed. Unfortunately
the
intensity of the emitted radiation varies with the
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FLUORESCENCE SPECTROSCOPY
wavelength throughout the entire range.
Since the
intensity of fluorescence is proportional to the excitation
radiation, thus
emission
upon using xenon lamp, variation of
intensity occurs not due to sample nature or
change of concentration but due to variation in source
intensity leading to erroneous results. To compensate for
the variation in source intensity and also for any other
instrumental variations, a double-beam instrument can be
used. The following figure illustrates a block diagram of
this type. A portion of the excitation energy is directed to
a solution of a fluorescent standard, its fluorescence is
proportional to intensity of incident radiation, and a
reference
signal
with
which
the
sample
signal
is
compared, is recorded.
2-Wavelength selector:
Two filters (both 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.
4-Detectors and readout meter:
Photomultiplier type is used since the intensity of
emitted radiation is small. Digital or analog or nullpoint
meter are used.
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FLUORESCENCE SPECTROSCOPY
Schematic diagram of an instrument to measure
fluorescence radiation
Schematic diagram of a typical spectrofluorometer
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FLUORESCENCE SPECTROSCOPY
Application of Fluorimetry:
Compounds which are intrinsically fluorescent are
easily determined at very low concentration by simple
fluorimetric method, for example, phenobarbitone, quinine,
emetine, adrenaline, cinchonine, reserpine vitamin A,
riboflavine………and many other
pharmaceuticals
and
natural products. Also, nonfluorescent substances can be
determined after chemical reaction.
Inorganic ions can be determined either by formation
of fluorescent chelates upon reaction with fluorimetric
reagents e.g. 8- hydroxyquinoline
(for Al), benzoin (for
Zn) or flavanol (for Zr) or by measuring the quenching of
fluorescence of a fluorescent substance in presence of
some ions.
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