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