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 What factors control whether a molecule will fluoresce?
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
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
Effect of Concentration on Fluorescence • 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
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 samples @ 77K to prevent collisional deactivation
Applications of Fluorescence • Fluorescence and phosphorescence are one of the most sensitive techniques available!