Fluorescence Lifetimes kf ki Pulse of Excited light fluorophore w/10ns decay fluorophore w/2ns decay
Fluorescence F Absorption Fluorescence Emission Higher Energy 400 500 600 700 Higher Energy Longer Wavelength Short Wavelength Excitation Long Wavelength Emission Absorption Fluorescence Emission INTENSITY 400 450 500 550 600 650 WAVELENGTH (nm)
Fluorescence Emission is Sensitive to Solvent Conditions More polar conditions generally yield a RED SHIFT in emission Less polar conditions usually result in a BLUE SHIFT Increasing Solvent Polarity INTENSITY 400 450 500 550 600 650 WAVELENGTH (nm) 400 500 600 700 Longer Wavelength Higher Energy
Solvent Effects
Solvent Relaxation
Solvent Relaxation of ANS
Quinine Sulfate in 0%, 25%, 50% and 75% EtOH Intensity Change Wavelength Shift
Lippert Equation
Lifetime t0 = 1/G t = 1/(G + k) Quantum Yield Q = t0/ t Intensity Iem = Q·f(l)·Iex ·(l) ·C·Z
Quantum Yield Measurements
Inner Filter Effects
Fluorescence Instrumentation