Fluorescence Spectroscopy L5

• Crystal Structure


  • Arrangement of atoms
  • Crystal size, lattice parameters
• Morphology


  • Porosity
  • Particle size and shape
• Chemical Composition


  • Types of elements present
• Luminescence spectroscopy


  Absorption First Followed by emission of photons in all the direction at lower frequencies

  
• Types of luminescence


  • Chemiluminescence
  • Phosphorescence
  • Fluorescence

  
• Photoluminescence


  Collectively Fluorescence and Phosphoresces

  
• Chemiluminescence


  Emission of light from an excited species as a result of a chemical rection

  
• Fluorescence


  Emission of light by a substance that has absorbed light or other electromagnetic radiation

  
• Fluorescence process


  • Molecule is excited from ground to excited state due to photon absorption
  • Excited state is unstable – decays back to ground state, emitting light in the process (fluorescence)

  
• Examples of fluorophores


  • Ethidium bromide (EtBr)
  • Phosphor
  • Fluorescein
  • Jellyfish Green fluorescent proteins
• Green fluorescent protein (GFP)


  • Small and inert molecule, can be fused to any protein for imaging
  • Widespread applications in biology., imaging, transgenics
• Emission spectrum


  Recorded by measuring the intensity of emitted radiation as a function of the emission wavelength

  
• Emission spectrum


  • Absorption of a photon of energy by a fluorophore, is an all or none phenomenon
  • Can only occur with incident light within the absorption range (strongest @ excitation maximum = peak in absorbance curve)
  • Temperature variation induces modification of global and local motions of the fluorophore environment and of the fluorophore itself, modifying its fluorescence emission feature
  • The intensity, position of the emission wavelength, and lifetime are some of the observables that will characterize a fluorophore

  
• Quantum Efficiency


  • Fluorescence Quantum efficiency (f) is the ratio of the # photons absorbed versus the # of emitted fluorescent photons. It is measured from 0 to 1 or in % (0to 100%)
  • It gives the probability of the excited state being deactivated by fluorescence rather than by another, non-radiative mechanism

  
• Common Names of Instruments

  • Fluorometer/Fluorimeter (using Filter)
  • Fluorescence Spectrometer/Spectrophotometer (using Monochromators)
• Fluorescence spectrometer


  • Light source
  • Excitation Monochromator
  • Sample
  • Detection Monochromator
  • Photomultiplicator

  
• Fluorescence is viewed at 90o Orientation

  
• Concentration and Fluorescence Intensity

  • Fluorescence intensity, F is proportional to the radiant power of the excitation beam absorbed by the species able to undergo fluorescence
  • F= k f (I0-I) where I0 is the power incident on the sample, I is the power after it traverses a length b of the solution, k is a constant depending on experimental factors, f fluorescence quantum efficiency

  
• Beer's law


  • I/I0=10-ebc and I=I0 10-ebc
  • F= k f I0 (1- 10-ebc)
  • F is not linear with concentration (Unlike Beer's Law)
  • F= k f I0 ebc, Fluorescence intensity is α to concentration
• Jablonski diagrams


  Classically presents the various energy levels involved in the absorption and emission of light by a fluorophore

  
• Jablonski diagrams


  • Straight arrows = absorption or emission of a photon (instantaneous process)
  • Wavy arrows = molecular internal conversion or non-radiative relaxation process (long timescales)
  • Thicker lines = electronic energy levels (S0, S1… Sn)
  • Thinner lines = vibrational energy states (0,1,2,3 etc)

  
• Franck-Condon energy diagrams


  • In the excited state, the electron is promoted to an anti-bonding orbital
  • Atoms in the bond are less tightly held (so potential energy curve shift to the right for S1)
• Overview of fluorescence process

  1. Excitation

  2. Non-radiative processes

  3. Emission

  
• Absorption of energy takes place on a time scale (10-15 s)

  
• Excitation lifetime


  Duration at which the molecule stays at the excited states S1 or S2
• Energy diagrams


  Graphical representation of energy levels and transitions

  
• In the excited state, the electron is promoted to an anti-bonding orbital
• Atoms in the bond are less tightly held (so potential energy curve shift to the right for S1)
• Fluorescence process


  • Excitation
  • Non-radiative processes
  • Emission
• Excitation


  1. Absorption of energy takes place on a time scale (10-15 s)

  2. Molecule is excited to higher vibrational energy level of S1 or S2

  
• Non-radiative processes


  1. Molecule relaxes to the lowest vibrational energy level of the first excited state S1(0)

  2. Internal conversion (loss of energy in the absence of light emission) occurs within 10–12 s or less

  
• As electronic energy increases, the energy levels grow more closely spaced
• The energy gap between S1 and S0 is significantly larger compared to other adjacent states, hence S1 lifetime is longer and radiative emission can compete effectively with non-radiative emission
• Emission


  1. Molecule relaxes to ground state S0

  2. Emission of photon (of lower energy than absorbed photon) => fluorescence

  3. Fluorescence occurs at a time scale of 10-8 s-tens of nanoseconds regime

  
• Singlet ground state (S0)

  Two electrons per orbital; electrons have opposite spin and are paired
• Singlet excited state (S1, S2 … Sn)


  One electron per orbital; electron in higher energy orbitals has the opposite spin orientation relative to electron in the lower orbital
• Triplet excited state (T1, T2…Tn)


  One electron per orbital; the excited valence electron may spontaneously reverse its spin (spin flip) so that electrons in both orbitals now have same spin orientation
• Intersystem crossing


  Non-radiative transition between states of different multiplicity via inversion of the spin of the excited electron

  
• Transitions between states of different multiplicity are in principle forbidden (e.g., S0 → T1,2)
• T1 → S0 is also forbidden without emission, instead phosphorescence occurs as molecule returns from T1 to S0 on a long time scale of 10-3-102 s