Molecular Spectroscopy
  2. Spectroscopy based on Emission
     Spectrofluorometry
 At the end of this lecture, you should be familiar with the following:
  • Fluorescence
  • Phosphorescence
  • Fluorophore
  • Singlet excited state
  • Triplet excited state
  • Fluorescence Quenching
  • Inner-filter effect
  • Quantum yield (Φf)
  • Fluorometer
  • Spectrofluorometer
  • Low pressure mercury vapor lamp
  • High-pressure Xe arc lamp
     Principles of fluorescence
  • Fluorometric analysis is extremely sensitive and is 
   used widely in many disciplines.
  • When a molecule absorbs electromagnetic energy, 
   this energy is usually lost as heat, as the molecule is 
   deactivated via collisional processes. 
  • Some molecules lose only part of the energy via 
   collisions, and then the electron drops back to the 
   ground state by emitting a photon of lower energy 
   (longer wavelength) than was absorbed. 
  • This phenomenon is called fluorescence; an 
   estimated 5 to 10% of all molecules fluoresce, 
   especially when excited by energetic UV radiation.
  • Absorbing a photon promotes one of the 
   electrons to a singlet excited state.
  • Emission of a photon from a singlet excited 
   state to a singlet ground state, or between any 
   two energy levels with the same spin, is called 
   fluorescence. 
  • In some cases an electron in a singlet excited 
   state is transformed to a triplet excited state in 
   which its spin is no longer paired with that of 
   the ground state.