Transcript Fluorescence, Quenching, and Applications Thereof

Fluorescence,
Quenching, and
Applications Thereof
Christopher Hampton,
Dr. E. F. Healy, Advisor
The Nature of Luminescence
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Production of light is most commonly
associated with heat
“Cold light” Phenomena (Rendell)
Fluorescence
 Phosphorescence
 Chemiluminescence
 Radioluminescence
 Triboluminescence
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Fluorescence
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Dilute atomic vapors
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Na 3s -> 3p*
589.6nm & 589.0nm
resonant fluorescence
Stokes Shift
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Molecular
fluorescence
Longer wavelengths
than the resonance
line
Jablonski Diagram
Absorbance and Emission
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Absorbance of photon
10^-14 to 10^-15s
Deactivation
Processes
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Vibrational Relaxation
Internal Conversion
External Conversion
Intersystem Crossing
Radiative Emission
Quantum Yield
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The ratio of the number of molecules that
luminesce to the total number of excited
molecules
Ф = Kf / Kf + Kisc + Kec + Kic + Kpred +
Kd
More efficient in * ->  than * -> n
Molar absorptivity 100x more for pp*
 Lifetime is shorter (10^-7 to 10^9) for pp*
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Quenching
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Internal Filtering* or Concentration
Quenching
Static Quenching (A*+Q -> AQ* ~> AQ)
Dynamic Quenching (A*+Q -> A+Q*)
Thermal Quenching
Oxygen Quenching
Photodecomposition or Reaction
Fluorescence Work
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Lucigenin has been
known to be
quenched by Chloride
anions for a long time
This phenomenon
has not been
extensively studied,
and no analytical
characterization has
been done in 40
years.
What They Did
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Lucigenin (dimethylbis(acridinium) nitrate)
was obtained and recrystallized twice
Absorption spectra were taken with a Cary
14
Fluorescence spectra Xenon ™ Corp 31A
nanosecond fluoremetry system.
Other organics used as shipped (DMSO,
Acetonitrile, and DMF )
Their Results
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KF (100%) - 17.4 eV
NaClO4 (100%) - ??
Na2SO4 (60%) - ??
NaC2H3O2 (31%) - 10.35
NaHSO3 (8.5%) - ??
KCN (5.5%) - 13.7
KCl (5.1%) - 13.0
Ns2SO3 (3.4%) - ??
NaSCN (1.4%) - ??
Na2S (0.3%) - 10.5
Hand-waving Lies and
Propaganda
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Linear dependence of
ionization potential
works if you leave out
1/2 of their data
points.
“Heavy atom” effect is
ignored for everything
but Cl, I, and Br…?
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No solvent effect on
quenching
Presence of amines
causes
photodecomposition
so can’t be studied
Fluoroscopy
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Benefits
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Small samples (3mL)
Widely available
Limitations
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Not all materials
fluoresce
Cost of fluorescent
materials
Detection limits
Fluoroscopy Experimental
Method
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Instrument Used
Chemicals Used
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All of ACS reagent grade
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Chloride solutions were made from a volumetric
NaCl standard solution obtained from Sigma
Aldrich (1g Cl- / 100g water)
All solutions were made with Millipore water
Experimental Method, contd.
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Standards prepared
Lucigenin
concentration from an
ethanol stock, diluted
in water
Solutions were
combined in a capped
cuvette, and vortexed
for 30-45 seconds
Experimental Method, contd.
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An excitation
spectrum was
obtained at 505nm
Maximum peak
intensity and
differentiation was
consistently observed
at 368 and 432 nm
0.1 µM Lucigenin Quenching
0.1 µM Lucigenin Regression
0.05 µM Lucigenin Quenching
0.05 µM Lucigenin Regression
What Does it Mean?
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50 nano-molar
concentration of
Lucigenin, and a 50
micro-molar Clsolutions.
1000:1 ratio of Cl- to
Lucigenin.
This can be further
reduced, but with an
introduction of noise
to signal ratio loss
Ok, but is it real?
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Still working at
concentrations that
are showing very
distinct patterns
We are approaching
the limits of detection
of our instrument and
operator
Contamination of our
water?
Where are we going from
here?
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CE is going to make
or break it.
Buffer has been
problematic
Repeat of 25 nm
fluorescence data set
(clean it up some)
Lower the Cl- :
Lucigenin ratio to
1:100 (I.e. 10-1µM Cl)
Questions?