Transcript The method of Electron Paramagnetic Resonance spectroscopy

Methods in the Department of Biological Sciences
Electron Paramagnetic Resonance
Dima Svistunenko
• EPR is a method of detection of
paramagnetic species
• What is paramagnetic?
• What is spin of a particle?
– Electric charge, its movement, magnetic field,
solenoid, small charged particle has a magnetic
field, spin, the small magnet.
• What is spin of a molecule?
– Many electrons, their spin adds up.
Paramagnetic species =
(S = 0)
Diamagnetic species =
Free radicals
Ions of transition metals
Other
(S = 0)
Dia + e- = Para
Para - e
-
= Dia
Dia - e- = Para
Para + e
-
= Dia
Paramagnetic species
=
(magnet)
• Why can we detect something
that is paramagnetic? How does
EPR works?
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•
•
•
•
energy splitting;
magnets in external magnetic field;
microwave radiation;
resonance formula;
• EPR spectrum of paramagnetic
species depends:
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– on the nature of the species
– on the instrumental conditions
(spectrometer type and settings)
Energy
How does EPR work?
Energy
How does EPR work?
DE = gb H1
DE
H1
External magnetic field
DE
hn
H1
H0
H2
External magnetic field
microwave
source
Energy
How does EPR work?
DE = gb H
DE
hn
gbH0 = hn
H1
H0
H2
External magnetic field
back
microwave
source
Energy
How does EPR work?
DE = gb H
•The instrument
•the photo;
•Microwave radiation and resonator;
–Radars, Cosmic Microwave Background radiation; microwave oven;
waveguides
•absorbance spectrum and first derivative;
•modulation amplitude;
•microwave frequency,
•RNR spectra at 2 diff frequencies;
•magnetic field window,
•microwave power;
•temperature of measurement
- liquid He or N2 vs room
back
Number of lines we see depends on the
setting for the modulation amplitude
Am = 3 G
Am = 2 G
Am = 0.2 G
back
EPR spectrum lineshape depends
on microwave frequency used
back
EPR spectrum intensity depends
on microwave power applied
back
EPR spectrum depends on
temperature
back
Paramagnetic species of different
nature give different EPR lineshapes
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Lineshapes to
simulate
• g-factor;
• hyperfine structure;
• linewidth
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• Temperature dependence
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• Biological samples spectra
examples
EPR spectrum intensity depends on
microwave power applied
back
The EPR spectra of human blood and muscle
measured at 10 K (that is –263oC)
The free radical in blood is formed in the reaction of
metHb with H2O2 which is continuously formed from .O2formed in turn during haemoglobin autoxidation
More than one radical:
how can we deconvolute
the spectrum into
components?
Tyr radical EPR spectra in different proteins
reported by different authors
Phenoxyl group in Tyr radical can rotate.
Hyperfine interaction of the spin with b-protons depends
on this rotation
McConnell, 1958:
B’ ~ 0
{1}
B’’ = 58 G
If we know
Ab1 and Ab2,
we can find
q and r
Trp14
Horse heart Mb
Trp14-OO.
peroxyl radical
Tyr103
Tyr103.
septet
Tyr146.
singlet
Tyr146