Molecular Fluorescence/Phosphorescence
Chapter 15 Section A mainly, a little of Sections B&C, not Section D.
Like atomic emission, the signal measured from molecular fluorescence
is emission of light from the sample. Thus in both cases a prerequisite is
an analyte in an excited state.
Atomic emission – heat it up to 2000K – 8000K. Molecules cannot
withstand these conditions. Excitation for molecular fluorescence and
phosphorescence from photon absorption (photoluminescence).
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The relative rates of these various processes are important.
Process
Rate (s)
Absorption
10-15
Vibrational Relaxation
10-12
Fluorescence
10-8
Phosphorescence
> 10-4
Internal/External Conversion
Variable
Note: The rate of vibrational relaxation >> rate of
fluorescence/phosphorescence.
Therefore: fluorescence/phosphorescence always occurs from the lowest
vibrational level of an excited state.
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Energy of fluorescence ≤ Energy of absorption
λ of fluorescence/phosphorescence ≥ λ of absorption
Two types of fluorescence/phosphorescence spectra can be obtained:
Excitation spectrum
Emission spectrum
How does one decide on the instrumental conditions (i.e. excitation,
emission wavelengths) when acquiring a fluorescence/phosphorescence
spectrum?
1.
Obtain an absorbance spectrum. Why?
2.
Fix excitation monochromator to top of most intense absorbance
and scan the emission monochromator to get an emission
spectrum. Why?
3.
Fix emission monochromator at wavelength of maximum
emission, scan the excitation monochromator to get an excitation
spectrum. Why?
4.
Fix excitation monochromator at wavelength of maximum
emission for excitation spectrum and obtain emission spectrum.
Why?
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This 4-step process is not foolproof, but reasonable. Alternatively obtain
emission spectra at all excitation wavelengths defined by absorbance
spectrum.
For most molecules excitation (absorption) does not result in emission
(luminescence). Most molecules have a low quantum yield. Structural
features which enhance the probability of fluorescence, or increase the
quantum yield, include aromaticity and structural rigidity. These factors
decrease the rate of radiationless deactivation processes such as internal
and external conversion, allowing luminescence to occur.
Quantum Yield = Φ =
Since Φ = 0 for most molecules, luminescence spectrometry is highly
selective (derivitization), but not generally applicable like absorption
spectrometry.
Molecular luminescence spectrometry is similar to atomic emission in 2
ways:
1) Very low detection limits due to the way the signal is generated
2) The signal is proportional to the number of excited state
molecules.
How does analyte concentration effect fluorescence intensity?
The luminescence intensity is proportional to the incident source power
to get the most excitation. Equivalent to temperature in atomic emission.
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A high intensity arc
source is used in
luminescence
spectrometry, but a
lower intensity W
lamp is used in
absorption
spectrometry since
the signal is not
proportional to
source output in
absorbance
spectrometry.
According to the above equation the signal intensity is logarithmically
proportional to concentration. But if the absorbance (ЄbC) < 0.05, then
1 – 10-ЄbC ~ ЄbC
So in dilute solution, the luminescence signal intensity = PoЄbCΦ = kC
For example, if Є = 104 (high but not uncommon), then what is the
maximum possible linear concentration, assuming a 1 cm pathlength
cell?
It is strange but at higher concentrations the fluorescence intensity can
decrease.
Common causes for this include selfquenching and self-absorption.
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To compare molecular UV-Vis absorption to molecular luminescence
spectrometry
UV-Vis
Fluorescence
Detection Limits (M)
10-7 – 10-8
10-11 – 10-12
Selectivity
Low
High
The low selectivity for UV-Vis means it is more generally applicable,
but interferences are a larger problem.
Chapter 15 Questions/Problems
1-3, 5, 9, 13
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