Introduction to Electron Spin Resonance Spectroscopy Techniques and Applications

An Introduction to Electron Spin Resonance (ESR)
Boris Dzikovski, ACERT, Cornell University
The application field

The basic ESR experiment.
Some theoretical background.
Nitroxide spin labels.
Some examples for extraction of parameters of
molecular dynamics from ESR spectra
Site directed spin labeling (SDSL)
ESR distance measurements
An introduction to Electron Spin Resonance (ESR), November 7, 2007
ESR is a spectroscopic technique that detects

chemical species that have unpaired electrons :
Transition metal ions and complexes Mn2+, Cu2+, Gd 3+ etc.

Simple inorganic compounds: O2 , NO, NO2 .
Short-lived intermediate radicals OH, H, F etc. in kinetics study
Defects in crystals
Electrons trapped in radiation damaged sites

Stable organic radicals
Triplet states
Biological applications:
Paramagnetic cofactors: iron sulfur, copper proteins
Free radicals of biological origin and their spin-trapping products
Spin-labeling
Energy-level diagram for two spin states as a function of applied field H.

This represents the simplest ESR transition (e.g.,
free electrons).
Allowed EPR transitions occur when Ms = 1
(Ms is the magnetic spin quantum number of the
spin state).
The equation describing the absorption (or
emission) of microwave energy between two spin
states is
E h g H
where:
E is the energy difference between the two spin

states
h is Plancks constant
is the microwave frequency
g is the Zeeman splitting factor (2.0023 for free
electron)
is the Bohr magneton
H is the applied magnetic field.
Relaxation
Evolution of a spin system is described
by Bloch equations:
Mx, My Mz magnetization components in the
rotating frame
0= eH0 the Larmor Frequency
e 1.76 10 7 rad /( s G )
T1- spin-lattice or longitudinal relaxation time

T2- spin-spin or traverse relaxation time
When properly integrated, the Bloch equations will yield the X', Y', and Z
components of magnetization as a function of time.
Stationary solution in the rotating frame gives a lorentzian line F ( H )
Gaussian line F ( H )
ESR linewidth:
T2
1
1 2T22 ( H H 0 ) 2
T2
1
exp( 2T22 ( H H 0 ) 2 ) = inhomogeneous broadening
2
2
1
k e H
T2'
1
1
1
T2' T2 2T1
k=1
Lorentz
k ln 2 Gauss
ESR and NMR are very different methods!

electron
proton
ratio
Rest mass
me =9.1094*10-28 g
mp =1.6726*10-24 g
5.446*10-4
Charge
e=-4.80286*10-10ESU
e=4.80286*10-10ESU
-1
Angular momentum
h/4
h/4
Magnetic dipole
moment
S=-ge eS
ge= 2.002322
S=-gN NS
gN= 5.5856
me=eh/4mec =
mN=eh/4mNc =
9.274*10-21 erg/G
5.0504*10-24 erg/G
1836.12
Frequency: Factor 1000 larger in EPR ! (GHz instead of MHz)

Coupling strength: Factor 1 000 000 larger in EPR ! (MHz instead of Hz)
Relaxation Times: Factor 1000 000 smaller in EPR ! (ns instead of ms)
= much higher techniqual requirements, but unique sensitivity to molecular motion
Sensitivity : Factor 1 000 000 better than in NMR !! (1nM instead of 1mM )
An ideal case, though
The Basic ESR Experiment (conventional ESR)

Source
Circulator
electromagnet
Modulation coils
Detector
Resonator (cavity)
The Basic ESR Experiment (conventional ESR)

Unlike NMR a large proportion of machines are still 'cw'.
That is they do not use pulsed detection methods
ESR is done from 1 to 300+GHz [30mT-10T or 30cm-1mm], up to 2000+ GHz
Machines are classified according to their source frequency :
Commonly used X-band at 9.5 GHz
L (1.5), S (3.0), C (6.0), Ku (17), K (23), Q (36), V (50), W (95), D(140), G(180)
Field modulation is used to encode the spectrum [1st derivative lineshape]
Use microwave transmission lines
Do spectroscopy with a few microwatts to a few milliwatts of power
Solid state [Gunn diode or DRO] or tube [klystron] sources
Temperatures from 4K (heme and non-heme iron) to 310K+ (in vivo/vitro)
Sensitivity : Increases as (frequency)2, but limited by sample size, field
homogeneity and component construction problems.
Practically (at X-band): detect 1011 spins,
a detectable concentration of ~10-9M.
Field modulation
A commercial 9GHz system
Commercial 95GHz
Spectrometer (3T)
The g-factor:
E = h = gH
The field at each spin influenced by local magnetic fields, not just the external field :
Heft = H + Hlocal so Heff = (1-s)H = (g/ge)H

-This field is induced by H, and so depends on the external field H
-g is an effective Zeeman factor, shifted from the electron g-factor, ge
-The shift in g is akin to the chemical shift of NMR
-The local induced field comes from the orbital motion of electrons, spin-orbit
coupling mixes J, L and S and shifts g, the shift can can be g<2 or g>2.
g is thus characteristic of different electronic structures and is also known as
the
Land splitting factor:
Light atoms, i.e.'organic' and first row transition metals with a single unpaired electron
can have g close to 2.0
Heavier atoms, and molecules or atoms with more than one unpaired electron can
have g-values very different from 2
g-values for some biologically important paramagnetic compounds
g-value
Flavin semiquinone, ubiquinone,
ascorbate, etc
2.0030 2.0050
Nitroxide spin labels and traps
2.0020 2.0090
sulphur radicals : S-S, S-H
2.02 - 2.06
MoV (in aldehyde oxidase)
1.94
Cu2+
2.0 - 2.4
Fe3+ (low spin)
1.4 - 3.1
Fe3+ (high spin)
2.0 - 10
A - the hyperfine splitting

The unpaired electron, which gives us the EPR spectrum,
is very sensitive to local fields in its surroundings.
Local fields arising from magnetic nuclei are
permanent and independent of H.
Interaction with neighboring nuclear magnetic dipoles gives the
nuclear hyperfine interaction and hyperfine splitting A

Corresponds to the NMR coupling constant J
A splittings are independent of the external field.
For several equivalent nuclei n, (2nMIM + 1) transitions are
observed for a nucleus M with a spin I
The relative intensities are given by Pascal's triangle for I =
1
1 1
1 2 1
1 3 3 1
1 4 6 4 1
1 5 10 10 5 1
1 6 15 20 15 6 1
1 7 21 35 35 21 7 1
I=1/2, 2I+1=2
I=1, 2I+1=3
Organic radicals in the liquid phase

Cyclooctatetraen anion
Butadien ion in liquid NH3
Pyrazine anion
Na+ is the counterion
Observation of the
1:8:28:56:70:56:28:8:1
spectrum shows that eight
protons are equivalent
Two sets of equivalent

protons: 2 and 4
K+ is the counterion
The figures are taken from the textbook by Wertz&Bolton
Anisotropy in g and A
Many measurements are made in the solid state in EPR spectroscopy.
The ability of EPR to obtain useful information from amorphous (glassy) and
polycrystalline (powders) as well as from single crystal materials has attracted
much biology and biochemistry research
Usually : gX, gY, gZ are not all equal, so g is anisotropic. Same for AX, AY, AZ.
For EPR the local symmetry at an unpaired electron center is
categorised as :
Cubic. If x = y = z is cubic (cubal, octahedral, tetrahedral) No
anisotropy in g and A.
Uniaxial (Axial). If x = y, and z is unique.
Linear rotation symmetry (at least 3-fold). Two principal values
each for g and A. For an arbitrary orientation:
g2 g 2 sin 2 g II2 cos 2
Rhombic. Three unequal components for g and A

For an arbitrary orientation:
2
2
2
g 2 g XX
sin 2 cos 2 gYY
sin 2 sin 2 g ZZ
cos 2
gx=2.0091, gy=2.0061, gz=2.0023

The field shift between the X- and Z- orientations is
H=h/gx- h/gz hg/4~11G
gx=2.0091, gy=2.0061, gz=2.0023

I=1/2, Ax= 6.2, Ay = 6.3, Az=33.6
gx=2.0091, gy=2.0061, gz=2.0023

I=1, Ax= 6.2, Ay = 6.3, Az=33.6
Powder and glass spectra

S=1/2, I=0, gx=gy<>gz Axially symmetric g-factor
Hr
h
h 2
[ g II cos 2 g 2 sin 2 ]1/ 2

g eff
is the angle between a given symmetry axis and the

magnetic field direction
The given solid angle is defined to be the ratio of the surface area A to the
total surface area on the sphere: A/4r2
sin
d2r2sind4r2sind
P ( H )dH sin d P ( H )
( g II2 cos 2 g 2 sin 2 ) 3 / 2

P( H )
h
( g II2 g 2 ) cos
1
P( H )
h H r3 ( g II2 g 2 ) cos
dH / d
Axial Lineshape
Rhombic Lineshape
EPR Middle-Earth
ESR signals around us

Toothpaste
Human hair
Q-band ESR spectrum of molecular

oxygen at reduced pressure
EPR dosimetry:
5000
10000
15000 G
The rotational angular momentum,
which is quenched in the liquid or
solid phases couples strongly to the
electronic spin and orbital angular
momenta
Determination of the accumulated radiation

dose by ESR of tooth enamel
Nitroxide spin labels

CH3
CH3
N
O
CH3
CH3
N
O
H3C
H3C
O
N
O
The g- and A-tensor frame for a

nitroxide radical
gx=2.0091, gy=2.0061, gz=2.0023

I=1, Ax= 6.2, Ay = 6.3, Az=33.6
9.4 GHz
Angular averaging in the case of S=1/2, I=1, gx=2.0089,

gy=2.0061, gz=2.0027, Ax= 5.2, Ay = 5.2, Az=31.0, X-Band
component I=+1
is spread over A- hg/4
component I=0
is spread over hg/4
component -1
is spread over A+ hg/4
The figures are from the monograph by Kuznetsov
High field EPR spectroscopy is the g-resolved spectroscopy,

the regions corresponding different orientations of the magnetic
axis relative to the external magnetic field do
not overlap
gx=2.0091, gy=2.0061, gz=2.0023

I=1, Ax= 6.2, Ay = 6.3, Az=33.6
170 GHz
Nitroxyl tumbling correlation time

As the molecule tumbles, the smaller splitting for mI = 0 is averaged more effectively
than the larger splittings, which causes differences in the linewidths of the three
hyperfine lines:
Nitroxyl Lineshapes
As the tumbling correlation time
decreases, the extent of averaging of
anisotropic features increases and the
spectrum approaches the 3-line signal
that is characteristic of rapid tumbling.
In the motional narrowing region, the
dependence of the width of an
individual hyperfine line on the nuclear
spin state (mI) can be expressed as
B (mI ) A B mI C m I
Rotation correlation times between
10-11 and 10-6 are detectable by ESR
X-band
Calculation of tumbling times in the case of fast isotropic

Thetumbling
parameters B and C are related to peak-to-peak amplitudes, I(m I) by:
1
B
2
I(0)
I ( 0)
I( 1)
I(1)
1
C
2
I(0)
I(0)
2
I(1)
I(1)
The high-field line has mI = -1.

Tumbling correlation times are calculated from B and C using
1
1
2
2 b
1
2 4Bo b 1
and C
B
3
g
15
o

o
Bo
3g o
Where
1
g o (g x g y g z )
3
( g z 0.5( g x g y ))

Bo
g o
2
(A z 0.5(A x A y ))
3
Bo is the peak-to-peak width of the center line

Hyperfine values (A) are in radians/s
The calculation assumes isotropic tumbling
Sample Calculation
OH
4-OH-TEMPO (tempol) in 9:1 glycerol:water

gx = 2.0094, gy = 2.0059, gz = 2.0023
N
O
Ax = 218x106, Ay = 222.5x106, Az = 2103x106 rad/s

I(+1) = 13.5, I(0) = 16.4, I(-1) = 3.4
(arbitrary units)
Bo = 3.52 Gauss
= 9.274x10-21 erg/G
= 9.2449x109 s-1
h=6.626x10-27 erg s
= 2.1x10-9 s from B or = 2.3x10-9 s from C
The disagreement is an indication of the approximate nature of this
calculation.
Determination of microviscosity:
V
kT
(Stocks-Einstein)
Extremely useful in oversaturated/overcooled disperse systems.

Example: testing photographic materials
g- and A- tensors are sensitive to the local polarity

gx
gy
gz
giso
Ax Ay Az
OH
N
O
Toluene
2.00986
Water/glyc 2.00878
Aiso
2.00626 2.00222 2.00617 6.2 7.0 34.3 15.6

2.00604 2.00215 2.00565 6.9 7.9 37.4 17.4
TEMPO in Emulsion: toluene/SDS/water
TEMPO in the LQ phase of DLPC

Partially A-resolved
g-resolved spectra
Orientational resolution of HF ESR for nitroxide spin labels

One of the main virtues of HF ESR over ESR at conventional microwave frequency is the
excellent orientational resolution for nitroxide spin labels. At HF, once motion is discernable in
the spectrum, one can discern about which axis the motion occurs.
Spin labeled fatty acids in solid cyclodextrins
NO
O
OH
NO
C
O
OH
O
C O
NO
Z-rotation vs. slow motion
Averaging effective tensor

components
(gxx+ gyy)/2, (gxx+ gyy)/2, gzz
(Axx+ Ayy)/2, (Axx+ Ayy)/2 Azz
Averaging real tensor components

gxx, gyy, gzz, Axx, Ayy, Azz
X-rotation
components
gxx,, (gyy+ gzz)/2, (gyy+ gzz)/2,
Axx, (Ayy+ Azz)/2, (Ayy+ Azz)/2
Diffusion tilt angle
Y-rotation
components
(gxx+ gzz)/2, gyy, (gxx+ gzz)/2,
(Axx+ Azz)/2, Ayy, (Axx+ Azz)/2
Lipid spin labels

DPPTC
16-PC
CH3
_
CH2
CH2
CH2
ym
zR
_
O
ym
CH3
H3C
CH2
CH3
CH3
C
CSL
xm
+N
O
P
CH2
zm
xm
CH2
Side view
zm
O
N
O
Upper view
O
CH
CH2
CH2
zd
O
N
O
zd
ym
xm
zR
N
O
Z-ordering
X-ordering
Y-ordering
ESR is one of the most powerful tools in lipid research

Phase state of lipids
Interaction of lipid with proteins, formation of lipoproteids, boundary lipid
etc.
Domains in model and biological membranes
Diffusion studies in the membrane phase
Polarity profiles in membranes
Membrane permeation profiles for oxygen and paramagnetic ions
ESR on aligned membranes

Spin-labeled gramicidin A in DPPC, 220 C
Aligned membrane
Vesicles
Simulation of angular dependent spectra is much freer of ambiguity, compared to

vesicles
Application of aligned membranes allows extracting information on relative
orientation of diffusion and magnetic axes, which can not be obtained from vesicles
Spin labeled Gramicidin A in DPPC at 170 GHz
4 5
9 0
M O M D
6 0 5 0 0
6 0 6 0 0
6 0 7 0 0
6 0 8 0 0
6 0 9 0 0
6 1 0 0 0
, G
6 1 1 0 0
MOMD: microscopic order macroscopic disorder.

An important case in biology
All orientations of the membrane normal relative to the magnetic field
are averaged in vesicles:
gx=2.0091, gy=2.0061, gz=2.0023
I=1, Ax= 6.2, Ay = 6.3, Az=33.6
N
O
9 GHz
170 GHz
OH
NO
NO
For a macroscopically disordered sample the orientation of the nitroxide

moiety manifests itself as a result of anisotropic molecular motion
around the principal axis of the molecular frame
Spin labeling. Peptides and proteins

Nitroxides are introduced into proteins as reporter groups to provide information
about local environment, overall tumbling rate of the protein or/and segmental mobility,
accessibility of the labeling site for polar/non-polar molecules, distance measurements
to other spin labels, co-factors, membrane surface.
O
O
R
OH
DCC
HO
N O
Labeling of the hydroxyl group
O
O
MTSL spin label is cysteine specific.
Protein
SH
NO
NO
CH3
SO2
CH2
Protein
CH2
SDSL = site directed spin labeling is introducing cysteines into the

protein
molecule by point mutations with following MTSL labeling.
Cysteine mapping of the protein molecule.
T4 lysozyme an example of successful EPR mapping
Multifrequency approach: high field EPR (250 GHz) spectra are

insensitive to the slow overall tumbling motion of the protein and
indicative of the internal motion
The 9 GHz spectra significantly affected by the overall tumbling and
less sensitive to the internal dynamics.
Multifrequency Analysis
Time scale
R (s-1)
Note: R = (6R)-1
Oxygen Accessibility
Oxygen accessibility and
probe mobility were measured
as a function of sequence
number for spin labels
attached to T4 lysozyme (T4L)
and cellular retinol binding
protein (CRBP).
The correlation between the
two parameters indicates that
the most mobile sites are also
the most oxygen accessible.
The repeat period of about 3.6
for T4L is consistent with the
-helical structure of this
segment of the protein.
W. L. Hubbell, H. S. Mchaourab, C. Altenbach, and M. A. Lietzow,
Structure 4, 779-783 (1996).
ESR distance measurements

Dipole-dipole interaction between two spins: Wdipolar
H DD
S S 3( S j rjk )( Sk rjk )
g j g j ( j 3 k
)
rjk
rjk5
2
B
H DD
1 2 3( 1 r )( 2 r )
3
r
r5
1 2 2
2
g
(
3
cos
1)
e
B
3
rjk
Proportional to (interspin distance)-3 angular dependent splitting.

Averaging over all orientations gives the Pake Doublet:
Some modern pulse EPR techniques (DQC, DEER) can cancel out all interactions
resulting in an EPR spectrum except the dipole interaction in spin pairs
Example: spin labeled Gramicidin A
2 e / r 3
5.2 10 4
dipolar [ MHz ] / 2
3
r []
Interspin distance=
30.9
dipolar, MHz,
DQC-EPR ruler:
10
20
30
40
50
60
70
80
90
100
DQC RULER
Location of mobile domains in a protein complex

using DQC-ESR:

Introduction to Electron Spin Resonance Spectroscopy Techniques and Applications

Caricato da

Informazioni sul documento

Descrizione originale:

Titolo originale

Copyright

Formati disponibili

Condividi questo documento

Condividi o incorpora il documento

Opzioni di condivisione

Hai trovato utile questo documento?

Questo contenuto è inappropriato?

Copyright:

Formati disponibili

Introduction to Electron Spin Resonance Spectroscopy Techniques and Applications

Caricato da

Copyright:

Formati disponibili

An Introduction to Electron Spin Resonance (ESR)

Boris Dzikovski, ACERT, Cornell University

The application field

ESR is a spectroscopic technique that detects

Transition metal ions and complexes Mn2+, Cu2+, Gd 3+ etc.

Electrons trapped in radiation damaged sites

Energy-level diagram for two spin states as a function of applied field H.

E is the energy difference between the two spin

An introduction to Electron Spin Resonance (ESR), November 7, 2007

T1- spin-lattice or longitudinal relaxation time

An introduction to Electron Spin Resonance (ESR), November 7, 2007

ESR and NMR are very different methods!

Frequency: Factor 1000 larger in EPR ! (GHz instead of MHz)

The Basic ESR Experiment (conventional ESR)

An introduction to Electron Spin Resonance (ESR), November 7, 2007

The Basic ESR Experiment (conventional ESR)

An introduction to Electron Spin Resonance (ESR), November 7, 2007

An introduction to Electron Spin Resonance (ESR), November 7, 2007

A commercial 9GHz system

An introduction to Electron Spin Resonance (ESR), November 7, 2007

An introduction to Electron Spin Resonance (ESR), November 7, 2007

Heft = H + Hlocal so Heff = (1-s)H = (g/ge)H

g-values for some biologically important paramagnetic compounds

Nitroxide spin labels and traps

sulphur radicals : S-S, S-H

MoV (in aldehyde oxidase)

Fe3+ (low spin)

Fe3+ (high spin)

An introduction to Electron Spin Resonance (ESR), November 7, 2007

A - the hyperfine splitting

nuclear hyperfine interaction and hyperfine splitting A

An introduction to Electron Spin Resonance (ESR), November 7, 2007

Organic radicals in the liquid phase

Butadien ion in liquid NH3

Na+ is the counterion

Two sets of equivalent

The figures are taken from the textbook by Wertz&Bolton

An introduction to Electron Spin Resonance (ESR), November 7, 2007

g2 g 2 sin 2 g II2 cos 2

Rhombic. Three unequal components for g and A

An introduction to Electron Spin Resonance (ESR), November 7, 2007

gx=2.0091, gy=2.0061, gz=2.0023

H=h/gx- h/gz hg/4~11G

An introduction to Electron Spin Resonance (ESR), November 7, 2007

gx=2.0091, gy=2.0061, gz=2.0023

An introduction to Electron Spin Resonance (ESR), November 7, 2007

gx=2.0091, gy=2.0061, gz=2.0023

An introduction to Electron Spin Resonance (ESR), November 7, 2007

Powder and glass spectra

[ g II cos 2 g 2 sin 2 ]1/ 2

is the angle between a given symmetry axis and the

( g II2 cos 2 g 2 sin 2 ) 3 / 2

An introduction to Electron Spin Resonance (ESR), November 7, 2007

An introduction to Electron Spin Resonance (ESR), November 7, 2007

ESR signals around us

Q-band ESR spectrum of molecular

Determination of the accumulated radiation

An introduction to Electron Spin Resonance (ESR), November 7, 2007

Nitroxide spin labels

The g- and A-tensor frame for a

An introduction to Electron Spin Resonance (ESR), November 7, 2007

gx=2.0091, gy=2.0061, gz=2.0023