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Fluorescence - PowerPoint PPT Presentation


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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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topics
Topics
  • Definition
  • Instrumentation
  • Sensitivity (nM-pM)
    • Contrast UV-vis measurements
  • Derivatization
  • Laser-induced Fluorescence
molecular fluorescence

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

competing rates
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
parameters
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

molecules that fluoresce
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
Instruments
  • Fluorimeter
  • Spectrophorimeter
fluorimeter
Fluorimeter

cuvette

Low pass

filter

High pass

filter

Photomultiplier

tube

Tungsten

source

spectrofluorimeter
Spectrofluorimeter

Emission Monochrometer

Excitation Monochrometer

slit

slit

slit

cuvette

Tungsten

source

Photomultiplier

tube

experiment with a spectroflurimeter
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
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