Fluorescence Spectroscopy

What factors control whether a molecule will fluoresce?

A. Quantum Yield

= ________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 fluorescence4.) 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’Po2.303bc

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!