1. • Quantum yield of fluorescence, Ff, is defined as
In practice, is measured by comparative measurements with reference
compound for which has been determined with high degree of accuracy.
Fluorescein
• Ideally, reference compound should have
– the same absorbance as the compound of interest at given excitation wavelength
– similar excitation-emission characteristics to compound of interest
– Same solvent, because intensity of emitted light is dependent on refractive index
(otherwise, apply correction
absorbedphotonsofnumber
emittedphotonsofnumber
F f
)(
)(
2
2
sn
un
I
I
s
f
u
f
s
f
u
f
F
F
1a. Quantum yield of fluorescence
2. Another definition for Ff is
where kr is the radiative rate constant and Sk is the sum of the rate
constants for all processes that depopulate the S1 state.
In the absence of competing pathways Ff=1
The observed fluorescence lifetime, is the average time the
molecule spends in the excited state, and it is
F
k
kr
f
k
f
1
Fluorescence lifetime
3. Illumination source
Broadband (Xe lamp)
Monochromatic (LED, laser)
Light delivery to sample
Lenses/mirrors
Optical fibers
Wavelength separation (potentially for both excitation and
emission)
Monochromator
Spectrograph
Detector
PMT
CCD camera
5. Phosphorescence:
T1 S1 S0 + hn
Intersystem crossing
Because intersystem crossing is a “forbidden” process which occurs
with low intensity with low probability phosphorescence is a slow
process where photons may be emitted over a period of fractions of
seconds to hours
•Phosphoresence – return from a triplet excited state
to a ground state; electron requires change in spin
orientation
6. The sequence leading
to phosphorescence:
The intersystem crossing
is the key step, where
T1 S1.
The triplet state acts
as a slowly leaking
reservoir, in that this
transition is normally
spin-forbidden
T1
S1
S0
T1 S1
Phosphorescence:
7. * Phosphorence spectra occure at longer wave length
Long life time, (10-6 to 102 Sec
Phosphoresence is not routinly obsereved at room temp.
(quenching of triplet excited state by oxygen)
Degasing is required
Several methods have been used to enable the observation of
phosphorescence,
One of the most commontechniques is to supercool solutions to a
rigid glass state usually at the temperature of liquid nitrogen (77 K).
At these low temperatures, molecular
collisions are greatly reduced and strong phosphorescence signals
are observed.
Due to long life-times, the molecule has a very high probability
of losing its excess energy by radiativeless routes such as internal
conversion, bimolecular collision, and photodecompositions
8. Inorganic salts and oxides of rare earths, europium, and Uranium
show phosphoresence
Compounds exhibit phosphorescence when adsorbed onto
certain substrates such as paper, cellulose, silica, etc.
*Phosphoroscope
*Two types of Phosphoroscopes
1) Rotating disc
2) Rotating can Phosphoroscope
9. *Measurement of Phosphoresence
*Samples are prepared in rigid media (polymer matrix)
*Temperature (liquid nitrogen)
• good solubility of the analyte
• formation of a clear rigid glass at 77 K
• low phosphorescence background (high
purity)
Polar compounds, ethanol is an excellent solvent and small
quantities of base or acid may be added to produce a clear solid.
For non-polar compounds, the most popular solvent is a
mixture of diethyl ether, isopentane and ethanol in the ratioof 5 :
5 : 2 respectively, called EPA
