Fluorescence Spectroscopy

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  • Created by: Rscottqub
  • Created on: 28-11-19 16:49
Fluorescence
ability to absorb light of specific wavelength and then ability to emit the light at a longer WL after a brief interval
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3 steps to fluorescence
1. Excitation 2. Vibration relaxation and internal conversion 3. Emission
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Excitation
Excitation of electrons by incoming photon to higher excited electronic state. Most electrons at room temp occupy lowest level of ground state
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Vibrational relaxation and internal conversion
Relaxation of excited electrons to the lowest level of the the 1st excited state . slightly slower than excitation
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Vibrational relaxation
Transfer of electrons from higher vibrational level to another within the same state
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Internal conversion
transfer of electrons from higher vibrational level to lower level between different states
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Emission
the emission of a longer WL photon - return of excited electron to ANY level of the ground state . Much slower than step 1/2
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Jablonski diagram - see diagram in notes
there are a no of ways of returning back to the ground state - intersystem crossing is possible but is rare as it is v slow
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intersystem crossing
transition between states of different spin. ie. excited triplet--> ground singlet
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Phosphorescence
radiative transition from excited triplet to ground singlet state
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We produce a flourescent spectrum by..
exciting a fluorophore with a specific WL
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Fluorophore
molecule which will exhibit fluorescence - contain multiple C=C conjugations
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Mirror image rule
Vib energy is similar to ground and excited states -spectrum looks like mirror image
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exception to mirror image rule
excitation of high energy photons - to higher levels that will then will quickly lose energy as it relaxes back down to ground state
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Instrumentation- monochromator
select specific WL, filters out all other WL of light
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Why apparatus set up at right angles
to increase sensitivity of measurement , collecting at 90 degrees reduces interference
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The ideal flurophore ...
high extinction coeff. (measure of light absorb capacity ) high fluorescence quantum yield. Large stokes shift
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Stokes shift
Difference between peak excitation WL and peak emission WL
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Interfering factors (5)
1. Quenching 2. Concentration 3. Temp 4. pH 5. inner filter effects
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Quenching
Decreases activity due to interaction between excited flurophore and surroundings
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Concentration
higher solute concentration means intensity of excitation light is not uniform. Also emitted light is reabsorbed by the other molecules
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Temp
Quantim efficiency is decreased as temp increases . Higher no of collisions decreases solvent viscosity
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pH
WL and emission id different for protonated an unprotected form of the fluorophore
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Inner Filter effects
decreased quantum yield due to reabsorb of emitted light or absorption of incident light by contaminants
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Advantages
highly sensitive , specific
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Cons
limited to fluorescent molecules , temp dependent , possible interference e
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Other cards in this set

Card 2

Front

3 steps to fluorescence

Back

1. Excitation 2. Vibration relaxation and internal conversion 3. Emission

Card 3

Front

Excitation

Back

Preview of the front of card 3

Card 4

Front

Vibrational relaxation and internal conversion

Back

Preview of the front of card 4

Card 5

Front

Vibrational relaxation

Back

Preview of the front of card 5
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