EPR1

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• EPR1
• Outline
• What is the Electron Paramagnetic Resonance
• g factor and hyper-fine splitting constant
  fingerprints of a radical
• Sensitivity to orientation
• Sensitivity to motion
• Sensitivity to neighbors
• Sensitivity to environment

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Electron paramagnetic resonance (EPR)
spectroscopy electron spin in external magnetic
field
, free electron
, Bohr magneton
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Spectroscopies. Energy of EPR transition
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Electron paramagnetic resonance (EPR)
spectroscopy how does it work?
In optics we measure sample response
(absorption/emission) directly
In EPR we measure a cavity detuning due to
resonant absorption by the sample
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Energy level scheme and transitions for free
electron in magnetic field
E
Electronmagnetic field interaction
ms 1/2,
, free electron
ms -1/2,
H0
0
EPR signal of single electron in external
magnetic field
H0
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g factor
g-factor reflects symmetry of electron density
z
z
y
y
x
x
Isotropic g factor, scalar value
Anisotropic g factor, tensor value
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g factor g 2.0058
How does EPR spectrum look like?
What would be a position of the spectrum, if
resonant frequency is 9.6 GHz?
what will be the line position at 96 GHz?
H0
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g factor gx2.0095, gy2.0058, gz2.0027
How does EPR spectrum look like?
What would be a position of the spectrum, if
resonant frequency is 9.6 GHz?
H
9
g factor gx2.0095, gy2.0058, gz2.0027,
random mixture
H
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g factor gx2.0095, gy2.0058, gz2.0027, rapid
motion, isotropic tumbling
How does EPR spectrum look like?
What would be a position of the spectrum, if
resonant frequency is 9.6 GHz?
What means rapid?
Coefficient of rotational diffusion
Isotropic tumbling correlation time
H
EPR spectrum reflects dynamics of paramagnetic
molecule
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Electron paramagnetic resonance (EPR)
spectroscopy nitroxide spin label
mS
mI
1
1/2
0
-1
-1
0
-1/2
1
nitroxide spin label three lines due to the
electron-nucleus hyperfine interaction
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Dependence of EPR lineshape on spin label
orientation
H0
y
Oriented system
x
Powder
EPR is sensitive to spin label orientation
relative to external magnetic field H0
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Dependence of EPR lineshape on spin label dynamics
H0
Rapid motion
What is rapid?
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Dependence of EPR lineshape on spin label dynamics
H0
Rapid motion
Slow motion
Slow restricted motion
EPR is sensitive to spin label dynamics
(correlation time and order parameter)
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Hyperfine interactions electron and proton,
rapid motion
ms
mI
1/2
Electronmagnetic field interaction
Electronnucleus interaction
1/2
E
-1/2
aH
-1/2
-1/2
1/2
aH
H
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Hyperfine interactions electron and 2 protons (I
1/2)
Free electron, S 1/2
a1
First proton, I 1/2
Second proton, I 1/2
a2
a2

• Questions
• a1a2
• Nitrogen I1
• Nitrogenproton, aN?aH
• Nitrogenproton, aNaH

H
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Spin trap (DMSO) radical. How many hyperfine
interactions can you find?
R -gt
H
R
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Spin trap (DMSO) radical. How many hyperfine
interactions can you find?
Interaction with nitrogen, aN14G
Interaction with hydrogen, aH12G
Interaction with hydrogen in OOH, aH0.5G line
broadening
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Spin trap (DMSO) radical. How many hyperfine
interactions can you find?
Interaction with nitrogen, aN14G
Interaction with one hydrogen, aH12G
Interaction with second hydrogen, aH12G
? ? ? ? ? ?
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Sensitivity of g and A to polarity of environment
Hydrogen bonding to the NO group of nitroxide
spin label reduces the g-tensor component
directed along the NO bond, and increases
hyperfine splitting value. These effects are
caused by decreased unpaired electron density on
the oxygen at hydrogen bonding.
Nitroxide spin label in polar environment
e-density shifts towards nitrogen, p-orbital
distortion
Isotropic magnetic parameters giso and Aiso.
Basic form of MTSL in 1 (toluene) 2
(acetonitrile) 3 (acetone) 4 (2-propanol) 5
(ethanol) 6 (water/ethanol, 37, v/v) and 7
(water/ethanol, 73, v/v) 8 (water buffered to
pH 6.0).
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A and g are sensitive to medium polarity to
resolve spectra for probes in water and lipid
TW
In water (polar) max A, min g In lipid, A is
less (decreased splitting) and g is slightly more
(whole spectrum shifts slightly to left). Add
the two spectra the high-field line is resolved.
Lineheights are proportional to mole fractions.
Use this to measure partitioning of probe between
lipid and water.
TL
hL
hw
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Thermal equilibrium, relaxation
Spin-lattice relaxation
Quenchers (paramagnetic ions in solution)
decrease T1 of a radical (lifetime of the excited
level) and increase relaxation rate from the
excited level
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Spin label accessibility to quencher power
saturation
Measure of accessibility to quencher
The difference between half-saturation powers ?
quencher reports on spin label accessibility to
quencher
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• Conclusions
• g-factor and hyper-fine splitting (A) tensors
  radical fingerprints
• EPR is sensitive to radical orientation in
  magnetic field
• EPR is sensitive to radical dynamics
• ERP is sensitive to local environment of the
  probe
• g-factor and hyper-fine splitting (A) are
  sensitive to polarity of environment