Fluorescence, Quenching, and Applications Thereof

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Fluorescence, Quenching, and Applications Thereof

• Christopher Hampton,
• Dr. E. F. Healy, Advisor

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The Nature of Luminescence

• Production of light is most commonly associated
  with heat
• Cold light Phenomena (Rendell)
• Fluorescence
• Phosphorescence
• Chemiluminescence
• Radioluminescence
• Triboluminescence

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Fluorescence

• Dilute atomic vapors
• Na 3s -gt 3p
• 589.6nm 589.0nm
• resonant fluorescence
• Stokes Shift
• Molecular fluorescence
• Longer wavelengths than the resonance line

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Jablonski Diagram
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Absorbance and Emission

• Absorbance of photon 10-14 to 10-15s
• Deactivation Processes
• Vibrational Relaxation
• Internal Conversion
• External Conversion
• Intersystem Crossing
• Radiative Emission

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Quantum Yield

• The ratio of the number of molecules that
  luminesce to the total number of excited
  molecules
• ? Kf / Kf Kisc Kec Kic Kpred Kd
• More efficient in ? -gt ? than ? -gt n
• Molar absorptivity 100x more for pp
• Lifetime is shorter (10-7 to 109) for pp

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Quenching

• Internal Filtering or Concentration Quenching
• Static Quenching (AQ -gt AQ gt AQ)
• Dynamic Quenching (AQ -gt AQ)
• Thermal Quenching
• Oxygen Quenching
• Photodecomposition or Reaction

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Fluorescence Work

• 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.

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What They Did

• 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 )

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Their Results

• 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

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Hand-waving Lies and Propaganda

• 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?

• No solvent effect on quenching
• Presence of amines causes photodecomposition so
  cant be studied

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Fluoroscopy

• Benefits
• Small samples (3mL)
• Widely available
• Limitations
• Not all materials fluoresce
• Cost of fluorescent materials
• Detection limits

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Fluoroscopy Experimental Method

• Instrument Used
• Chemicals Used
• All of ACS reagent grade
• 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

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Experimental Method, contd.

• Standards prepared
• Lucigenin concentration from an ethanol stock,
  diluted in water
• Solutions were combined in a capped cuvette, and
  vortexed for 30-45 seconds

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Experimental Method, contd.

• An excitation spectrum was obtained at 505nm
• Maximum peak intensity and differentiation was
  consistently observed at 368 and 432 nm

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0.1 µM Lucigenin Quenching
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0.1 µM Lucigenin Regression
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0.05 µM Lucigenin Quenching
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0.05 µM Lucigenin Regression
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What Does it Mean?

• 50 nano-molar concentration of Lucigenin, and a
  50 micro-molar Cl- solutions.
• 10001 ratio of Cl- to Lucigenin.
• This can be further reduced, but with an
  introduction of noise to signal ratio loss

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Ok, but is it real?

• 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?

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Where are we going from here?

• 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 1100 (I.e.
  10-1µM Cl-)

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Questions?