Molecular Luminescence Spectrometry

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Chapter 15

• Molecular Luminescence Spectrometry

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Molecular Fluorescence

• Optical emission from molecules that have been
  excited to higher energy levels by absorption of
  electromagnetic radiation.

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Photoluminescence
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Photoluminescence

• Light is directed onto a sample, where it is
  absorbed and imparts excess energy into the
  material in a process called "photo-excitation."
  One way this excess energy can be dissipated by
  the sample is through the emission of light, or
  luminescence.

• The intensity and spectral content of this
  photoluminescence is a direct measure of various
  important material properties.

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Photoluminescence

• Band gap determination. The most common radiative
  transition in semiconductors is between states in
  the conduction and valence bands, with the energy
  difference being known as the band gap.
• Recombination mechanisms. The return to
  equilibrium, also known as "recombination," can
  involve both radiative and nonradiative
  processes. The amount of photoluminescence and
  its dependence on the level of photo-excitation
  and temperature are directly related to the
  dominant recombination process.

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PHOTOLUMINESCENCE

• Fluorescence Does not involve change in
  electron
• spin short lived
  (less than microsecond). Can be observed at
• room temperature
  in solution.
• 2. Phosphorescence Involves change in electron
  spin.
• Long lived
  (seconds). Can be
• observed at
  low temperature in
• frozen or
  solid matrices.
• 3. Chemiluminescence Light emission due to a
  chemical reaction.

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Electron Spin

• The Pauli exclusion principle states that no two
  electrons in an atom can have the same set of
  four quantum numbers. This restriction requires
  that no more than two must have opposed spin
  states. Because of spin pairing, most molecules
  exhibit no net magnetic field and are thus said
  to be diamagnetic. In contrast, free radical,
  which contain unpaired electrons, have a magnetic
  moment are said to be paramagnetic.

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Singlet/Triplet States

• Singlet State A molecular electrons state in
  which all electron spins are paired is called a
  singlet state and no splitting of electronic
  energy levels occurs when the molecule is exposed
  to a magnetic field. Net spin S is zero. Spin
  Multiplicity 2S 1 1.
• Doublet State Free radical (due to odd
  electron). Net spin S is 1/2. Spin Multiplicity
  2S 1 2.

• Triplet State Electron Spins in the ground and
  excited electronic states are not paired. Net
  spin S 1. Spin multiplicity 2S 1 3.

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JABLONSKI DIAGRAM
Vibrational deactivation
Solid lines Radiative process Dashed
lines-Nonradiative Process
Intersystem Crossing (Ki)
Absorption
Fl (Kf)
Ph
Singlet state Quenching Kcc
quenching
Internal conversion Kic
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Rates of Absorption and Emission...

• The rate at which a photon of radiation is
  absorbed is enormous, the process requiring on
  the order o f 10-14 to 10-15s. Fluorescence
  emission, on the other hand, occurs at a
  significantly slower rate. Here, the lifetime of
  the excited state is inversely related to the
  molar absorptivity of the absorption peak
  corresponding to the excitation process.

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QUANTUM YIELD
Quantum yield or quantum efficiency (?) Quantum
yield for a fluorescent process is the ratio
of the number of molecules that fluoresce to the
total number of excited molecules. For a highly
flurorescent molecule such as fluorescein ? 1
and for a nonluminesceing molecule ? 0.

• can be defined in terms of the various rate
  constants
• In the Jablonski diagram as
• ? kf/(kf ki kcc kic)

Fluorescence (kf) Singlet State quenching (kcc)
Intersystem crossing to triplet state from
singlet state (ki) Internal conversion (kic)
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Idealized absorption and emission spectra
The 0-0 transition is common to both absorption
and emission. When these transitions overlap we
have resonance emission.
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ABSORPTION AND EMISSIOIN SPECTRA
In the absorption spectra transitions to higher
vibrational energies lead to absorptions at lower
wavelengths. In the emission spectrum transition
from the 0 vibrational level of the excited state
to higher vibrational levels of the ground state
lead to emissions at higher wavelengths. The
wavelength maxima for the absorption and
emission spectra under resonance conditions are
identical in accordance with the Franck-Condon
principle if the life time of the excited state
is very short.. In many systems the wavelength
maxima for the absorption and emission spectra
do not coincide due to Loss of energy of the
excited state by collision with solvent
molecules.
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ABSORPTION AND EMISSIOIN SPECTRA (CONTD)
Emission Spectrum Plot of the emission
intensity at 90o to the incident radiation as a
function of emission wavelength for a fixed
excitation wavelength. Excitation Spectrum
Plot of the emission intensity at a fixed
emission wavelength at 90o to the
incident radiation as a function of excitation
wavelength. Fluorescence lifetimes 10-9
10-6 s. Phosphorescence lifetimes 10-3
seconds.
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Sample Excitation and Emission Spectra
Excitation
Emission
Source Skoog, Holler, and Nieman, Principles of
Instrumental Analysis, 5th edition, Saunders
College Publishing.
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Sample Spectra Excitation (left), measure
luminescence at fixed wavelength while varying
excitation wavelength. Fluorescence (middle) and
phosphorescence (right), excitation is fixed and
record emission as function of wavelength.
Phosphorescence is susceptible to O2 and
collisions with solvent molecules. Triplet states
are rapidly deactivated under these conditions.
For many molecules phosphorescence can only be
observed at low temperatures in frozen matrices
in the absence of O2. (RTP application)
Source Skoog, Holler, and Nieman, Principles of
Instrumental Analysis, 5th edition, Saunders
College Publishing.
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FLUORESCENCE AND STRUCTURE
1. Fluorescence from singlet states of ?- ? have
more intensity than those from n- ? transitions
as the molar absorptivities for ?- ? absorptions
are much higher than those for n- ?
absorptions. 2. Simple heterocycles do not
exhibit fluorescence. The n-?singlet quickly
converts to the n- ? triplet and no Fluorescence
is observed.
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FLUORESCENCE AND STRUCTURE (CONTD)
3. Fusion of heterocycles to benzene rings
increases the molar absorptivity for n- ?
absorptions and shortens the life time of the n-
? singlet preventing its conversion to triplet.
This increases fluorescence quantum efficiency.
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STRUCTURAL RIGIDITY
Flurorescence is favored in molecules with
structural rigidity. The quantum yields for
fluorescence for fluorene and biphenyl are 1 and
0.2 respectively. The increased rigidity of
fluorene stabilizes the ?- ? singlet state
leading to higher quantum yield. Chelation also
can lead to increased fluorescnece.
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HEAVY ATOM EFFECT
A very significant influence on the fluroescence
quantum yield of the benzene ring which is due to
?- ? singlet states is observed with halogen
substitution. The quantum yield decreases with
the atomic number of the halogen. This called
the heavy atom effect. The probability for
intersystem crossing increases with increasing
atomic number of the halogen which
reduces fluorescence. Substitution of carboxylic
acid or carbonyl group on benzene generally
inhibits fluorescence due to the n- ? states
being lower in energy than the ?- ? states and
these do not fluoresce efficiently.
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TEMPERATURE AND SOLVENT EFFECTS

• Quantum yield of fluorescence of most molecules
  decreases with increasing temperature due to
  collisional deactivation of the singlet state.
• 2. Fluorescence is decreased by solvent
  containing heavy atoms such as as those
  containing halogens.
• Heavy atoms promote intersystem crossing to the
  triplet state. This decreases fluorescence
  quantum yield but increases phosphorescence
  quantum yield.
• 4. Solvent Viscosity lower viscosity, lower
  quantum yield.
• 5. Concentration
• Self-quenching due to collisions of excited
  molecules.
• Self-absorbance when fluorescence emission and
  absorbance
• wavelengths overlap.

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Effect of Concentration on Fluorescence Intensity
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Components of Fluorometers and Spectrofluorometers

• Sources A more intense source in needed than the
  tungsten of hydrogen lamp.
• Lamps The most common source for filter
  fluorometer is a low-pressure mercury vapor lamp
  equipped with a fused silica window. For
  spectrofluorometers, a 75 to 450-W high-pressure
  xenon arc lamp in commonly employed.
• Lasers Most commercial spectrofluorometers
  utilize lamp sources because they are less
  expensive and less troublesome to use.

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Components of Fluorometers and Spectrofluorometers

• Filters and Monochromators Both interface and
  absorption filters have been used in fluorometers
  for wavelength selection of both the excitation
  beam and the resulting fluorescence radiation.
  Most spectrofluorometers are equipped with at
  least one and sometimes two grating
  monochromators.
• Transducers Photomultiplier tubes are the most
  common transducers in sensitive fluorescence
  instruments.
• Cell and Cell Compartments Both cylindrical and
  rectangular cell fabricated of glass or silica
  are employed for fluorescence measurements.

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Fluorometer Schematic
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Fluorometer Figure
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Spectrofluormeter Figure
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ABSORBANCE VS. LUMINESCENCE
ABSORBANCE
LUMINESCENCE Most compounds absorb All
compounds that absorb do not emit light.
(good selectivity)
Narrow liner dynamic range Large
linear dynamic range Not very sensitive to
impurities Very sensitive to impurities, 13
order of magnitude better than in
absorption spectrometry Absorbance monitored
along Luminescence monitored the axis of the
incident at 90o to the
incident radiation
radiation
While emission occurs in all directions only the
photons emitted at 90o to the incident radiation
are monitored to avoid interference from
transmitted photons.
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