fluorescence lifetimes
DESCRIPTION
Pulse of Excited light fluorophore w/10ns decay fluorophore w/2ns decay. Fluorescence Lifetimes. k f. k i. 0. 400. 500. 600. 700. F. Short Wavelength Excitation. Long Wavelength Emission. Longer Wavelength. Higher Energy. INTENSITY. 400. 450. 500. 550. 600. 650. - PowerPoint PPT PresentationTRANSCRIPT
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