March 19, 2001

Fluorescence and Luminescence

Watch spelling: fl U-O rescence (not flOUr...)

• Also note Monochromator does not end in ...meter
• It's a device that monochromates light not a meter that measures something

Spectrophotometry

• monitors light passing through sample
• measure blank carefully
• measure small changes due to absorption
• if change < noise level, undetectable
• typically can measure millimolar solutions
• amplification useless-- S/N unchanged

• measures light emitted by sample
• blank is basically darkness or stray light
• weak signal can be amplified
• limit is typically dark signal of PMT
• signal must exceed dark current
• can often measure micromolar or nanomolar
• current detection limit is 1 atom per cubic meter
• however, need to excite electronic states
• ok for atoms-- flames, plasmas,...
• need different technique for molecules

• Fluorescence
  • specific-- excited by absorption of light and emission in short period (vague)
    • casual-- may often be used to apply to phosphorescence (fluorescent tubes, fluoroscopes)
    • name has origins in fluorite minerals which can be made to glow; name predate fluorine which is derived from the name of the mineral.
• Phosphorescence
  • specific-- excited by absorption of light and emission is slow (microsec to hours)
    • outside of chemistry-- broader and includes other excitation (like radioactivity)
• Luminescence
  • most general term--
    • includes fluorescence and phosphorescence
    • includes chemilumininescence and biological luminescence
    • include thermo- and electro-luminescence

(generally, refer to text and diagrams-- these notes lack diagrams and have less detail)

Some background

• Most molecules exist in singlet state
  • all electrons are paired
  • net spin is zero
  • 2S+1 = 1 (one state, a singlet)
  • we will often use the letter S to label a singlet state
    • (don't confuse with S=spin quantum number)
• this describes the electronic state
  • for an atom, that's all there is
  • for a molecule we can expect many different states (different vibrational states for the same electronic state)
    • vibrational energy << electronic energy
• absorption of light follows a selection rule
  • delta S = 0
  • so excited state is also a Singlet state
  • such allowed transitions are of high probability
  • so the reverse step is also very probably
  • singlet states will have very short lifetime
  • they fluoresce-- emit light and go the a lower state
    • of course, there may be competition and the energy may end up in some other route and not as fluorescence
• The atomic case is called Resonance Fluorescence
  • the wavelength of emitted light matches that of absorbed light
    • (some minor shifts due to Doppler and other line broadening)
    • (may also see other lines)
    • example-- UV excites to level 2
    • see emission from 2 to 0 (same wavelength as excitation)
    • also see some emission from 2 to 1 (less energy)
    • molecules in level 1 can still emit at the 1 to 0 transition
    • other two lines correspond to other absorptions you can observe
  • some atoms hit the wall , lose energy and don't fluoresce

• typically all molecules are in the electronic ground state
  • this is the lowest singlet state, we'll call it S^o
  • they are also usually in the lowest vibrational state as well
• they absorb light and are excited to an upper singlet (S) state, called S1
  • but there are many vibrational levels here so we see a broad absorption band
  • produce excited states in many vibrational levels
  • transitions require at least or more energy than the simple S^o -->S¹ difference
• in practice, loss of vibrational energy is very rapid
  • to solvent, to crystal lattice, to nearby molecules
  • this is a radiationless transition
  • result is all molecules in the same (ground) vibrational state or S¹
• now we see fluorescence from S¹ to S⁰
  • but there are many vibrational levels available in So
  • so we see a broad emission band
  • the most energetic transition would match the S⁰ --> S¹ energy
• as a result, the emission spectrum is less energetic than the excitation spectrum
  • we say the emission is red shifted
• since the vibrational levels of S¹ and S⁰ are similar
  • the shape of absorption and emission bands are similar
  • emission is often a mirror image of excitation or absorption spectrum
• The quantum yield can be nearly 1.0 (1 photon out per photon absorbed)
  • still, fluorescence is typically a weak signal
    • the emission is isotropic and most of it misses our detector system
    • we also need to limit absorption to about 15%
    • more than that and fluorescence is limited to thin layer of sample
• fluorescence is rapid (psec to nsec typically)
  • simple fluorometer , spectrofluorometer or fluorescence spectrometer ignore the timing

• Pro-- can be extremely sensitive (micro to nanomolar)
• Pro-- often has negligible interferences (few samples really fluoresce)
• Pro-- extra degree of control over other electronic spectra (both excitation and emission wavelengths are characteristic of the sample)
• Con-most samples don't fluoresce (can be a blessing too)
• Con- instrumentation is more expensive typically
  • needed larger mirrors and gratings for a weak signal
  • need two monochromators for best setup
  • need rather intense lamps (Xenon arc typical)
• Con-- subject to interferences like quenching

There is almost always a lower energy excited state than S1

• This is a triplet state (T) with two unpaired electrons
• It costs energy to pair electrons

• that's a forbidden transition
• occurs so weakly, it's seldom a significant part of the absorption spectrum

• this is also radiationless
• it does require a change in angular momentum, so it needs a partner
• partner could be solvent molecule, nearby molecule
• often called, generally, a lattice effect
• efficiency of the process varies with the species

• we lose fluorescence intensity
• can get fluorescence quantum yield to drop from 1.0 to 0.5, 0.1, 0.001 ...

• if to the ground singlet, it's still forbidden
• so process is very slow
• if intensity is spread out over long time, it's very weak

• quenching is a common case.