Spectrofluorimetry

Origin of Photoluminescence

Absorption of visible or UV radiation raises molecule to an excited state
Electron absorbs quantum of energy and jumps to a higher energy orbital.
When electron drops back to the ground state, excitation energy can be liberated by...

1. QUENCHING (or RADIATIONLESS TRANSFER)
   - most common
   Energy temporarily increases vibrational and rotational energy of bonds in the molecule - ultimately dissipated as heat in surrounding solvent.
2. RE-EMISSION OF RADIATION,
   - less common
   Gives rise to... FLUORESCENCE and/or PHOSPHORESCENCE
   (two forms of PHOTOLUMINESCENCE)

The energy transitions associated with photoluminescence can be represented on Morse energy surface diagrams.

Energy Changes in Fluorescence

The Morse curves (see above) display energy transitions for an electron, in a covalent bond A-B, excited by hn, then re-emitting the energy as photoluminescence

• Before excitation, molecules and bonds are mostly in the lowest vibrational level of the ground electronic state. Hence excitation (absorption of hn) begins from lowest level.
  But - for quantum mechanical reasons - excited electron enters a higher vibrational level in the excited state.
  
• In the excited state, the excess vibrational energy is rapidly lost.. So de-excitation (with emission of hn) takes place from the lowest vibrational level of excited state.
  But electron dropping back to the ground state re-enters it first at a higher vibrational level before losing the excess vibrational energy.

Stokes' Law Of Fluorescence

Net effect of energy transitions depicted and described above is:

As energy is inversely related to wavelength:

Fluorescence vs Phosphorescence

Usually re-emission of radiation, if it occurs, is within nanoseconds (10^-9 sec)of the excitation = FLUORESCENCE.

PHOSPHORESCENCE - much slower (and rarer) process.
After source of excitation radiation is switched off, phosphorescence emission continues for periods that vary from milliseconds to weeks.
Phosphorescence is caused by electron becoming transferred into a triplet state. (Electrons of the same spin in the one orbital). Triplet states have long lifetime so phosphoresence persists. Mostly requires low temperatures (<<0^oC) - at higher T, triplet state is de-activated by quenching.

Fluorescence (including "delayed fluorescence" (microseconds)) is the most analytically useful type of photoluminescence.

Instrumentation : The Spectrofluorimeter

A Fluorescent Species Has Three Spectra

Inter-relatedness of Fluorescence and Absorption Spectra

• Not every absorption peak gives rise to fluorescence.
  However peaks in a fluorescence excitation spectrum usually correspond closely in wavelength to absorption peaks.
  
• No readily predictable correlation between heights of the absorption peak and the excitation peak.
  Efficiency in converting the energy to fluorescence (vs quenching) differs for different absorption bands.
  
• Fluorescence emission spectrum has maximum at higher wavelength than excitation spectrum (Stokes' law).
  Emission spectrum is usually simpler - usually only a single broad peak.
  
• "Scattering peaks" when the wavelengths of primary and secondary monochromators coincide. (See small blips at l_f in the excitation and and l_e in emission spectrum) - artefacts (dust particles)

Reproducibility of Spectra

An ABSORPTION spectrum is reproducible when scanned on different instruments (Provided not distorted by inappropriate settings of scan speed, band pass width etc).
But FLUORESCENCE spectra (excitation and emission) are less reproducible; This is because Fluorescence is not measured relative to a blank.
Hence fluorescence spectra affected by:

1. How intensity of light source varies with wavelength
2. How response of photomultiplier detector varies with wavelength

Absorption spectra unaffected by the above because absorbance is based on ratio I_o/I. Instrument-related factors affect I_o and I in same proportion, so cancel.

Quantitative Spectrofluorimetry

Linear response, Fluorescence vs Mass of Analyte, only at LOW CONCENTRATIONS.
Important factor is the INNER FILTER EFFECT.

Advantages and Disadvantages of Spectrofluorimetric Assay

Major advantage is high sensitivity.
Capacity to assay at much lower concentrations than by absorption spectrophotometry.

A potential advantage is improved selectivity.
Requirement to set two wavelengths in spectrofluorimetry (excitation and emission) hence unlikely that an impurity is being co-measured - it would have to absorb and emit at the same two wavelengths.

Disadvantages are many. A very exacting technique, requiring careful attention to experimental detail, including purity of reagents.

What Compounds Fluoresce?

Structural requirements for fluorophore are more difficult to define than for chromophore.
But a molecule must first absorb hn to undergo the first step of electron excitation.
Then, because of Stokes' shift to higher wavelength for the emitted light, most fluorescent species are compounds that absorb UV light. For biological structures, an aromatic ring is the most common structural requirement. Additional requirement for fluorescence is that quenching must not occur while the molecule is in the excited state.In aromatic molecules, electron withdrawing groups tend to produce quenching. eg -NO₂, or -COOH substituents on the aromatic ring tend to decrease the chances of fluorescence. Conversely, electron rich groups inhibit quenching. eg -NH₂ or -OH are likely to enhance the fluorescence, e.g., Fluorimetric Assay Of Catecholamines. (Biologically active amines - include the hormones adrenalin and noradrenalin. Elevated levels in some medical conditions).

Product has 4 electron donor groups. Highly fluorescent at 530 nm (360 nm excitation). Basis of sensitive assay for adrenaline.