Chapter 27
Molecular Fluorescence Spectroscopy
Fluorescence is a photoluminescence process in
which atoms or molecules are excited by
absorption of electromagnetic radiation. The
excited species then relax to the ground state,
giving up their excess energy as photons. One of
the most attractive features of molecular
fluorescence is its inherent sensitivity, which is
often one to three orders of magnitude better than
absorption spectroscopy.
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Another advantage is the large linear
concentration range of fluorescence methods,
which is significantly greater than those
encountered
in
absorption
spectroscopy.
Fluorescence methods are, however, much less
widely applicable than absorption methods
because of the relatively limited number of
chemical systems that show appreciable
fluorescence.
Principles of Molecular Fluorescence
Molecular fluorescence is measured by exciting
the sample at the absorption wavelength, also
called excitation wavelength, and measuring the
emission at a longer wavelength called the
emission or fluorescence wavelength. Usually,
fluorescence emission is measured at right angles
to the incident beam so as to avoid measuring the
incident radiation. The short-lived emission that
occurs
is
called
fluorescence,
whereas
luminescence that is much longer lasting is called
phosphorescence.
Excitation Spectra and Fluorescence Spectra
Because the energy differences between
vibrational states is about the same for both
ground and excited states, the absorption, or
excitation spectrum, and the fluorescence
spectrum for a compound often appear as
approximate mirror images of one another with
overlap occurring near the origin transition (0
vibrational level of E1 to 0 vibrational level of E0).
There are many exceptions to this mirror-image
rule, particularly when the excited and ground
states have different molecular geometries or
when different fluorescence bands originate from
different parts of the molecule.
Concentration and Fluorescence Intensity
The radiant power of fluorescence F is
proportional to the radiant power of the
excitation beam absorbed:
F = K(P0 – P)
where, P0 is the radiant power of the beam
incident on the sample and P is the radiant power
after it traverses a pathlength b of the medium.
Constant K depends on the Quantum efficiency.
The efficiency of fluoresced to the number
absorbed.
At low concentrations where fluorescence is most often
employed
F = K’P0c
where, c is the concentration of the fluorescent species
and K’ is a new proportionality constant. F is directly
proportional to analyte concentration. Thus, a plot of the
fluorescent radiant power versus the concentration of the
emitting species should be, and ordinarily is, linear at low
concentrations. When c becomes great enough that the
absorbance is larger than about 0.05, linearity is lost and
F begins to reach a plateau with concentration. This effect
is known as a primary absorption inner filter effect. In
fact, at high concentrations, fluorescence radiant power
can even begin to decrease with increasing concentration.
Molecular Phosphorescence Spectroscopy
Phosphorescence is a photoluminescence
phenomenon that is quite similar to fluorescence.
Understanding the difference between these two
phenomena requires and understanding of
electron spins and the difference between a
singlet state and a triplet state. Ordinary
molecules exist in the ground state with their
electron spins paired. A molecular electronic
state in which all electron spins are paired is said
to be a singlet state.
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When one of a pair of electrons in a molecule is
excited to a higher-energy level, a singlet or a
triplet state can be produced. In the excited
singlet state the spin of the promoted electron is
still opposite that of the remaining electron. In
the triplet state, however, the spins of the two
electrons become unpaired and are thus parallel.
The excited triplet state is less energetic than the
corresponding excited singlet state.
Chemiluminescence Methods
Chemiluminescence is produced when a chemical
reaction yields an electronically excited molecule,
which emits light as it returns to the ground state.
Chemiluminescence reactions are encountered in a
number of biological systems, where the process is
often called bioluminescence.
One attractive feature of chemiluminescence for
analytical uses, is the very simple instrumentation.
Since no external source of radiation is needed for
excitation, the instrument may consist of only a
reaction vessel and a photomultiplier tube. Generally,
no wavelength selection device is needed because the
only source of radiant is the chemical reaction.
MOLECULAR SCATTERING METHODS
Several additional processes, however, can occur
when radiation interacts with matter. One of the
most important of these is scattering of
electromagnetic radiation. Scattering can be
divided into two classes: elastic scattering, in
which the scattered radiation is of the same
energy as the incident radiation, and inelastic
scattering, in which the scattered radiation has
higher or lower energy than the incident
radiation. Both of these types of phenomena have
useful analytical applications.
…continued…
Inelastic light-scattering methods have become
very important in recent years, particularly those
methods based on the Raman effect. Raman
spectroscopy involves inelastic scattering of
radiation caused by vibrational and rotational
transition. The Raman method is complementary
to IR spectroscopy and can be used to obtain
qualitative,
structural
and
quantitative
information about molecular species.
Chapter 28
ATOMIC SPECTROSCOPY
Atomic spectroscopy is used for the qualitative
and quantitative determination of 70 to 80
elements. Detection limits for many of these lie
in the sub-parts-per-million range. The methods
can be based on absorption, emission, or
fluorescence.
Atomic
absorption
(AA)
spectroscopy currently is the most widely used
of these techniques.
Atomic Absorption Spectroscopy
In AA spectroscopy, as in all atomic
spectroscopic methods, the sample must be
converted into an atomic vapor by a process
known as atomization. In this process, the
sample is volatilized and decomposed to
produce atoms and perhaps some ions in the gas
phase. Several methods are used to atomize
samples. The two most important of these for
AA spectroscopy are flame and furnace
atomization.
Flame Atomic Absorption Spectroscopy
The radiation of a line source of the element of
interest, typically a hollow cathode lamp, is
directed through the flame containing the atomic
vapor. Solution samples are usually brought into
the flame by means of a sprayer or nebulizer,
which produces small sample droplets. The
solvent from the droplets quickly vaporizes, and
the resulting salt particles vaporize and
decompose into atoms, ion and electrons.
…continued…
Atoms in the sample will absorb radiation
emitted by the same atom in the hollow cathode
lamp and thus attenuate the power of the source.
Usually a monochromator is used to separate a
spectral line of the element of interest from any
background radiation from the source or the
flame. A photomultiplier tube is typically used
to convert the radiant power from the source
into a related electrical current.
Furnace Atomic Absorption Spectroscopy
Furnace, or electrothermal, AA uses the same
instrumental setup as flame atomizer except that a
furnace atomizer is substituted for the burner.
With furnace AA, discrete amounts of sample,
usually microliter volumes, are deposited in the
furnace. A multistep heating program is usually
applied to desolvate the sample, ash, or char the
organic material present and then produce the
atomic vapor. Furnace AA gives a transient signal
that reaches a peak in a few seconds. Furnace AA
is usually one to two orders of magnitude more
sensitive than flame AA.