Excitation Spectrum

Excitation Spectrum Absorption Spectrum

1.0 1.0
Emission at 650 nm

0.8

Absorbance (O.D.)
0.8

0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0
300 400 500 600 300 400 500 600
Excitation Wavelength (nm) Wavelength (nm)

If emitting from a single species:

Excitation spectrum should match absorption
spectrum!
 Excited State Decay
S1 Rate equation:

dnS1
 k A nS0  (k r  k nr )nS1
Energy kA knr kr dt
dnS1/dt = 0

S0 Sample is illuminated with photons of constant

intensity, a steady-state concentration of S1 is
kA = excitation rate rapidly achieved.
kr = radiative rate nS 0 k A
nS1 
knr = non-radiative rate k F  k nr
Substitution for photon flux and the relationship
between kA and kr then (math happens):

kr
 
k nr  k r
# of photons emitted
= = 0 to 1
# of photons absorbed
 Need of finding the excited state life time of molecules

Effect of Con. of analyte on the

fluorescence spectrum:
PET

Effect of Con. of amine on the

fluorescence spectrum:
Dyes and Pigments, 2016, 134, 306
Quenching Mechanism
 Deactivation Pathways for S1 State

Fluorescence decay, non-radiative

decay, intersystem crossing, singlet-
mediated reactions

Rate of decay of S1 depends on kR, kNR, kISC and kD

Rate = - d[S1]/dt = (kF + kNR + kISC + kD) [S1]

The fluorescence lifetime  is defined as

 = 1/ (kF + kNR + kISC + kD)  ≠ 1/ kF

If (kNR + kISC + kD) = 0, then

 = 1/ kF = 0

0 is called natural or radiative lifetime

The fluorescence lifetime is generally equated with the
time required for the intensity to decay to 1/e of its initial
value
First order process
I(t) = I0e-t/
100
If t = ,
I(t) = I0e-1 = I0/e
N(t)
= 0.37 I0 or 50
I(t) 37


Lifetime is the time at which fluorescence intensity 0
reaches (1/e)th of its initial value time

 63% of N0 decay at t ≤ 

 Even after t = 2 or 3, few molecules remain in the excited state

Quenching Mechanism
Single and double exp. decay processes

Decrease of fluorescence life time on

addition of analyte
Additional Reading:

Section 4.19-4.28 (Page no. 200-230): Modern Molecular Photochemistry of

Organic Molecules: Nicholas J. Turro, J. C. Scaiano, V. Ramamurthy