Fluorescence Lifetimes
-
Upload
desiderio-hierro -
Category
Documents
-
view
32 -
download
0
description
Transcript of Fluorescence Lifetimes
Fluorescence Lifetimes
kf ki
Pulse of Excited light
fluorophore w/10ns decay
fluorophore w/2ns decay
F
Fluorescence
0 400 500 600 700
Higher EnergyLonger Wavelength
Short WavelengthExcitation
Long WavelengthEmission
FluorescenceEmission
INT
EN
SIT
Y
400 450 500 550 600 650WAVELENGTH (nm)
Absorption
FluorescenceEmission
Absorption
Fluorescence Emission is Sensitive to Solvent ConditionsIN
TE
NS
ITY
400 450 500 550 600 650WAVELENGTH (nm)
0 400 500 600 700
Higher EnergyLonger Wavelength
IncreasingSolvent Polarity
More polar conditions generallyyield a RED SHIFT in emission
Less polar conditions usuallyresult in a BLUE SHIFT
Solvent Effects
Solvent Relaxation
Solvent Relaxation of ANS
Quinine Sulfate in 0%, 25%, 50% and 75% EtOH
Intensity Change Wavelength Shift
Lippert Equation
.
.
12
1
12
1
.
.
.
).(
)(2
2
2
11
2
3
solventofindexrefractiven
solventofconstantdielectric
wheren
nlitypolarizabinalorientatiof
dipoleefluorophorofradiusa
statesgroundandexcitedofmomentsdipole
lossesenergylvibrationaofoncontributiC
cminwavenumber
where
Ca
f
GorE
GE
• Lifetime
• Quantum Yield
Q =
• Intensity
Iem = Qf()Iex C
2
R
R
RR n
n
OD
OD
I
IQQ
Quantum Yield Measurements
Inner Filter Effects
Fluorescence Instrumentation