Fluorescence

Topics Definition Instrumentation Sensitivity (nM-pM) –Contrast UV-vis measurements Derivatization Laser-induced Fluorescence

S0S0 S2S2 S1S1 T1T1 v2v2 v4v4 v1v1 v3v3 v5v5 v1v1 v3v3 v5v5 v1v1 v3v3 v5v5 v7v7 v1v1 v3v3 v5v5 v7v7 Absorption Vibrational relaxation Fluorescence Phosphorescence Internal conversion Intersystem crossing Molecular Fluorescence

Competing Rates Absorption occurs on the femptosecond time scale ( s) Relaxation from the ground state occurs through the fastest available process Most molecules do not fluoresce because the excited vibrational states of S 0 overlap with the S 1 state and relaxation can take place rapidly by vibrational relaxation Generally on the picosecond timescale

Parameters The intensity of the fluorescence is given by F = 2.3K′  bcP 0 K′ is directly proportional to  the quantum efficiency  = k f / (k f + k i + k ec + k ic + k pd + k d ), competing rates

Molecules that fluoresce Fluorescence- relaxation through the emission of a photon Generally occurs on the ms-ns timescale. Molecules that fluoresce tend to be rigid aromatic compounds that possess limited vibrational freedom Phosphorescence is the emission of a photon from a excited triplet state Phosphorescence is an extremely rare property

Instruments Fluorimeter Spectrophorimeter

Fluorimeter Tungsten source Low pass filter High pass filter cuvette Photomultiplier tube

Spectrofluorimeter Tungsten source Excitation Monochrometer Photomultiplier tube cuvette slit Emission Monochrometer

Spectra

Experiment with a spectroflurimeter Select a trial excitation wavelength Collect the emission spectrum (scan emission wavelength) Select the wavelength of max emission Collect the excitation spectra (scan excitation wavelength) Select wavelength of max excitation Perform experiment with wavelength of max emission and max excitation

Why fluorescence? More sensitive than UV/vis absorption by a factor of Measuring against a zero background Biochemical tool; Fluorescent tagging Laser Induced fluorescence –F is proportional to intensity of source –Selectivity of monochromatic light source –Convenient with HPLC