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Fluorescence, Quenching, and Applications Thereof

Fluorescence, Quenching, and Applications Thereof. Christopher Hampton, Dr. E. F. Healy, Advisor. The Nature of Luminescence. Production of light is most commonly associated with heat “Cold light” Phenomena (Rendell) Fluorescence Phosphorescence Chemiluminescence Radioluminescence

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Fluorescence, Quenching, and Applications Thereof

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  1. Fluorescence, Quenching, and Applications Thereof Christopher Hampton, Dr. E. F. Healy, Advisor

  2. The Nature of Luminescence • Production of light is most commonly associated with heat • “Cold light” Phenomena (Rendell) • Fluorescence • Phosphorescence • Chemiluminescence • Radioluminescence • Triboluminescence

  3. Fluorescence • Dilute atomic vapors • Na 3s -> 3p* • 589.6nm & 589.0nm • resonant fluorescence • Stokes Shift • Molecular fluorescence • Longer wavelengths than the resonance line

  4. Jablonski Diagram

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

  6. Quantum Yield • 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*

  7. Quenching • Internal Filtering* or Concentration Quenching • Static Quenching (A*+Q -> AQ* ~> AQ) • Dynamic Quenching (A*+Q -> A+Q*) • Thermal Quenching • Oxygen Quenching • Photodecomposition or Reaction

  8. 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.

  9. 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 )

  10. 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

  11. 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 can’t be studied Hand-waving Lies and Propaganda

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

  13. 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

  14. 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

  15. Experimental Method, contd. • An excitation spectrum was obtained at 505nm • Maximum peak intensity and differentiation was consistently observed at 368 and 432 nm

  16. 0.1 µM Lucigenin Quenching

  17. 0.1 µM Lucigenin Regression

  18. 0.05 µM Lucigenin Quenching

  19. 0.05 µM Lucigenin Regression

  20. What Does it Mean? • 50 nano-molar concentration of Lucigenin, and a 50 micro-molar Cl- solutions. • 1000:1 ratio of Cl- to Lucigenin. • This can be further reduced, but with an introduction of noise to signal ratio loss

  21. 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?

  22. 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 1:100 (I.e. 10-1µM Cl-)

  23. Questions?

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