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Fluorescence Spectroscopy
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What factors control whether a molecule will fluoresce?
Quantum Yield f = ________kf______________ kf + ki + kec + kic + kpd + kd kf = rate constant for fluorescence ki = “ “ for intersystem crossing kec = “ “ for external conversion kic = “ “ for internal conversion kpd = “ “ for pre-dissociation kd = “ “ for dissociation
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What factors control whether a molecule will fluoresce?
B. Structure: 1.) Aromatic 2.) Rigid structures exhibit more 3.) Heavy atoms will decrease fluorescence 4.) Fluorescence will increase when molecule is adhered to surface
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What factors control whether a molecule will fluoresce?
C. Temperature and Solvent Effects 1.) Lower temperature increases fluorescence 2.) Solvent contains heavy atoms will decrease fluorescence but increase phosphorescence
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Effect of Concentration on Fluorescence
F = k’Po2.303ebc F = Fluorescence k’ = constant depending on quantum efficiency Po = incident power For high concentrations linear relationship no longer holds due to self quenching What is self quenching? Excited molecules collide and radiationless transfer of energy occurs
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Instrument Design for Fluorimeters
Important considerations for fluorimeters 1. Must have intense source, D2 & W not intense enough so use Xe or Hg Fluorescence is not as strong as absorbtion so need more sensitive detectors like PMT 3. Cells transparent on 4 sides detection usually at right angles of source 4. Phosphorescence measurements may be made on fluorimeters w/ 2 differences Irradiation + time delay + measurement Cooled 77K to prevent collisional deactivation
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Applications of Fluorescence
Fluorescence and phosphorescence are one of the most sensitive techniques available!
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