Physical Biochemistry Fluorescence Spectroscopy [Page 2] 1. Absorption (should be 1 vertical arrow)2. Internal conversion3. Solvent relaxation
Excited state has different electrondistribution / electrostatic dipole
Solvent cage reorganises itself to better stabilise excited state
This influences the energy and lifetime of the excited state
Causes fluorescence to occur at a lower energy than expected 4. Fluorescence5. Internal conversionSolvent polarity and pH can influence the fluorescence spectra, however it is difficult to define clear cut rules on how this should occur, due to the short timescales.Fluorescence Parameters:Quantum efficiency (Q.E.): Emitted photons / Absorbed photons. If the molecule has been excited, what is the probability that it will emit a photo, rather than lose its energy through internal conversion. Values between 0-1.Molar extinction coefficient () [M
]: Absorbance of a 1 molar solution in a 1cm flightpath at a specified wavelength. Simply out, it is the probability that a molecule will undergo a transition from the lower state to an excited state. Wavelength dependent ± the closer you are to the energy difference, the better you excite the molecule.Brightness (sensitivity) = Q.E.
. Wavelength dependent due to . concentration must be kept constant.Changing the absorption wavelength will change the peak intensity of the spectra; the shape of the emission spectrawill stay the same.Absorption and fluorescence spectra are independent.Lifetime:Emission of a photon is based on probability, not a defined time. An average time for moleculeswaiting in the excited state is the lifetime of the molecule.