Photoluminescence Spectrometry
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.
Kinetics dictate whether luminescence occurs.
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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.
A spectrum of quinine illustrating internal conversion followed by
fluorescence emission:
Often excitation and emission spectra are mirror images of each other…
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Spectrofluorometers may be single channel or multichannel:
The multichannel instrument provides the capability to acquire a total
luminescence spectrum in a matter of seconds.
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 (ππ*) and structural rigidity
(minimize internal conversion/overlapping energy levels). These factors
decrease the rate of radiationless deactivation processes (i.e. internal
conversion), allowing luminescence to occur.
Quantum Yield = Φ =
5
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.
A high intensity Xe
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.
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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.
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.
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Single molecule detection
Chapter 15 Questions/Problems
1-6, 9, 13
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