Stern-Volmer experiments can be used to study photochemical reactions:
e.g. photoaddition reaction
O
CN kq
C=O * +
CN NC CN
acetone dicyanoethylene dicyanooxetane
fluorescent
non-fluorescent
-can detect fluorescence from acetone to monitor progress of reaction
-acetone fluorescence quenched by dicyanoethylene
Energy Transfer
M* + Q M + Q*
Fluorescence quenching - example of energy transfer.
Energy exchange occurs through short distances ~ 2nm (collisions).
However, energy can be transferred over longer distances than collision diameters.
In general:
D* + A D + A*
donor acceptor
-energy transfer can occur up to distances of 10 nm
FÖrster Transfer
-depends on overlap of the spectral bands of emission from donor
& absorption by the acceptor.
Fluorescence Resonance Energy Transfer (FRET)
• The excitation frequency of the DONOR must not excite the ACCEPTOR.!
• The emission spectrum of the DONOR must overlap the excitation spectrum of the ACCEPTOR.!
• The emission spectrum of the DONOR cannot significantly overlap with emission of ACCEPTOR. !
• DONOR and ACCEPTOR molecules must be in close proximity (typically 10–100 Å). !
For example, 2 solutions in benzene:
(a) 10-3M in anthracene (b) 10-3M in perylene
(a) anthracene
-excitation of either solution within the chromphore’s excitation band
produces fluorescence from the fluorophore.
e.g. excitation of anthracene solution at 340 nm gives relatively
intense fluorescence between ~ 370-470 nm.
Similarly, excitation of the perylene containing solution at 410 nm “see”
fluorescence from perylene (430-550 nm)
(b) perylene
(c)
Now, a solution containing both 10-3M anthracene and 10-3M perylene:
(i) excitation at 410 nm (where only perylene absorbs)
gives perylene fluorescence (unperturbed by presence of anthracene).
(ii) excitation at 340 nm (hardly absorbed at all by perylene) produces fluorescence
form both anthracene and perylene.
2 0 0
1 6 0
perylene
anthracene
Intensity
1 2 0
8 0
4 0
0
400 500
Wavelength / nm
However, anthracene fluorescence quenched in mixture.
FÖrster Transfer-form of non-radiative transfer
- depends upon spectral overlap of donor (anthracene) & absorption band of
acceptor (perylene)
Involves dipolar interactions:
-effectively electronic oscillations in D * induce oscillations in electron to be
promoted in A
(like resonance interactions between 2 tuning forks).
D* + A D + A*
Consequently, D * returns to D and A is promoted to A *
-also known as fluorescence resonance energy transfer (FRET).
This form of energy transfer is enhanced by:
(i) good overlap of fluorescence spectrum of D * & absorbance spectrum of A
(ii) large FF for D * (in absence of A)
(iii) large eA for A
Energy transfer Efficiency
FF
ET = 1- for D in presence of acceptor
F oF for D in absence of acceptor
transfer
efficiency
Ro6
E T=
Ro6 + R 6
distance between donor & acceptor
spectroscopic characteristic
of each donor/acceptor pair
- ET increases with decreasing distance (R)
Donor & acceptor must be within 10 nm (critical transfer distance)
-sometimes referred to as Spectroscopic Ruler Technique
-used extensively to measure distances in biological systems & across
science in general.
Applications of FRET in Chemistry & Biology
Example: rhodopsin -protein (primary absorber of light contained in the eye)
-allows vision to take place
rhodopsin- consists of an opsin protein
molecule attached to 11- cis retinal
-amino acid on the surface of rhodopsin was
labelled covalently with the energy
donor I AEDANS
Iodoacetyl amino ethyl amino naphthalene sulphonic acid (I AEDANS)
D A
FF of label decreased from 0.75 to 0.68 due to quenching by the cis retinal acceptor
0.68
ET = 1 – = 0.093
0.75
Ro6
Ro is 5.4 nm for this pair from E T=
Ro6 + R6
can calculate R
AEDANS (D)
7.9 nm
11- cis retinal (A)
opsin protein
rhodopsin
Can calculate the distance (R) between the surface of the rhodopsin protein
& cis retinal as 7.9 nm
• Example: Consider a study of the protein rhodopsin. When an amino acid
on the surface of rhodopsin was labelled covalently with the energy donor
1.5-I AEDANS (3), the fluorescence quantum yield of the label decreased
from 0.75 to 0.68 due to quenching by the visual pigment 11-cis-retinal (4).
Calculate the distance between the surface of the protein and 11-cis-
retinal (Ro = 5.4 nm for the 1.5-I AEDANS/11-cis-retinal pair )
