Fluorescence Spectroscopy
What factors control whether a
molecule will fluoresce?
A. Quantum Yield
f=
________kf______________
kf + ki + kec + kic + kpd + kd
kf = rate constant for
fluorescence
ki = “ “ for intersystem crossing
kec = “ “ for external conversion
kic = “ “ for internal conversion
kpd = “ “ for pre-dissociation
kd = “ “ for dissociation
What factors control whether a
molecule will fluoresce?
B. Structure:
1.) Aromatic
2.) Rigid structures exhibit more
3.) Heavy atoms will decrease fluorescence
4.) Fluorescence will increase when molecule is adhered
to surface
What factors control whether a
molecule will fluoresce?
C. Temperature and Solvent Effects
1.) Lower temperature increases fluorescence
2.) Solvent contains heavy atoms will decrease
fluorescence but increase phosphorescence
Effect of Concentration on
Fluorescence
• Effect of concentration on Fluorescence
– F = k’Po2.303ebc
F = Fluorescence
k’ = constant depending on quantum efficiency
Po = incident power
For high concentrations linear relationship no
longer holds due to self quenching
What is self quenching? Excited molecules collide and
radiationless transfer of energy occurs
Instrument Design for Fluorimeters
Important considerations for
fluorimeters
1. Must have intense source, D2
& W not intense enough so
use Xe or Hg
1. Fluorescence is not as
strong as absorbtion so
need more sensitive
detectors like PMT
2. 3. Cells transparent on 4
sides detection usually at
right angles of source
4. Phosphorescence
measurements may be
made on fluorimeters w/
2 differences
a. Irradiation + time delay +
measurement
b. Cooled samples @ 77K
to prevent collisional
deactivation
Applications of Fluorescence
• Fluorescence and
phosphorescence
are one of the most
sensitive
techniques
available!