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Fluorescence

Fluorescence. Topics. Definition Instrumentation Sensitivity (nM-pM) Contrast UV-vis measurements Derivatization Laser-induced Fluorescence. v 7. v 7. v 5. v 5. v 3. v 3. v 1. v 1. v 5. v 3. v 1. Molecular Fluorescence. Absorption Vibrational relaxation Fluorescence

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Fluorescence

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  1. Fluorescence

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

  3. v7 v7 v5 v5 v3 v3 v1 v1 v5 v3 v1 Molecular Fluorescence Absorption Vibrational relaxation Fluorescence Phosphorescence Internal conversion Intersystem crossing S2 v5 S1 v3 v1 T1 v4 v2 S0

  4. Competing Rates • Absorption occurs on the femptosecond time scale (10-15 s) • Relaxation from the ground state occurs through the fastest available process • Most molecules do not fluoresce because the excited vibrational states of S0 overlap with the S1 state and relaxation can take place rapidly by vibrational relaxation • Generally on the picosecond timescale

  5. Parameters The intensity of the fluorescence is given by F = 2.3K′ebcP0 K′ is directly proportional to f, the quantum efficiency F = kf / (kf + ki + kec + kic + kpd + kd), competing rates

  6. 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

  7. Instruments • Fluorimeter • Spectrophorimeter

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

  9. Spectrofluorimeter Emission Monochrometer Excitation Monochrometer slit slit slit cuvette Tungsten source Photomultiplier tube

  10. Spectra

  11. 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

  12. Why fluorescence? • More sensitive than UV/vis absorption by a factor of 10-105. • 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

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