179

10 EPR Spectroscopy
10.1 Introduction to EPR Spectroscopy
Electron paramagnetic resonance (EPR) shares the theoretical description and many
experimental concepts with NMR spectroscopy. From a methodological point of view, the
main difference is the much larger magnetic moment of electron spins, which exceeds the one
of protons by a factor of 660. Therefore EPR has a higher sensitivity at given spin
concentration, can observe spin-spin interactions over longer distances, and is sensitive to
molecular motion at shorter time scales. Resonance frequencies are in the microwave (mw)
range (see Table 10.1). Typical pulse lengths and signal observation times are also shorter by
about a factor of 500. Spectrometer technology is thus more complicated and may differ
considerably between the different mw bands. In this lecture course, we shall refer to the most
common band, the X band, unless noted otherwise.
Table 10.1: Microwave frequency bands used in EPR spectroscopy (band definition by Radio Society
of Great Britain)..
From an application point of view almost all pure substances contain magnetic nuclei
and are thus accessible to NMR spectroscopy, while only few pure substances contain
unpaired electrons and are thus accessible to EPR spectroscopy. This is because chemical
binding is based on electron pair formation with spin cancellation. Most stable compounds are
Band Frequency range
Typical EPR
Frequency
[GHz]
Typical
wavelength
[mm]
Typical
a
EPR
Field
[mT]
a. Assuming = 2.002319
L
S
X
K
u
Q
V
W
D
1-2
2-4
8-12
12-18
30-50
50-75
75-110
110-170
1.5
3.0
9.5
17
36
70
95
140
200
100
30
17
8
4
3
2
54
110
340
600
1280
2500
3390
5000
v
g g
e
~
B
0
180

thus diamagnetic. The occurence of an EPR signal is thus often related to enhanced reactivity,
as for instance in transition metal complexes and free radicals. Therefore, EPR is particularly
well suited for studies on synthetic and biological catalysts and for studies of radical-induced
degradation processes in organisms and materials.
As an example consider the enzyme methyl-coenzyme M reductase (MCR), which
catalyzes methane formation from methyl-coenzyme M (methyl-CoM) and coenzyme B.
Biochemical studies do not reveal whether MCR catalyzes the reaction by attacking the
thioether sulfur atom of methyl-CoM or the carbon atom of the CH
3
S group. This question was
addressed by studying the reaction of MCR with the inhibitor 3-bromopropane sulfonate
(BPS), which results in a stable paramagnetic species with a single electron in a orbital
on the nickel ion. By using BPS
13
C labeled at the methyl group and the two-dimensional
HYSCORE experiment at X band (Section 10.4.4.4), it was possible to detect hyperfine
coupling between the unpaired electron and the methyl carbon atom (Figure 10.1). The
couplings to the protons of BPS were obtained from Q-band HYSCORE and electron nuclear
double resonance (ENDOR) spectra. From this information a model for the binding mode
could be proposed.
Figure 10.1: Determination of the binding mode of the inhibitor 3-bromopropane sulfonate (BPS) to
the active center F430 of methyl-coenzyme M reductase (MCR). A) Structure of the active center. B)
Structure of the inhibitor. C) X-band HYSCORE spectrum for the product obtained from F430 and BPS
13
C-labelled at the -position. The left quadrant contains the
13
C peaks. D) Simulated HYSCORE
spectrum. E) Structural model for BPS bound to F430 (from Hinderberger D. et al, Angew. Chem. Int.
Ed. 2006, 45, 3602-3607).
Some defects in inorganic materials, such as semiconductors, are also paramagnetic.
These defects determine the electronic and optical properties of these materials. EPR
d
x
2
y
2

-30 -20 -10 0 10 20 30

0
10
20
30
-30 -20 -10 0 10 20 30
0
10
20
30
?
2
/
MHz
?
2 /
MHz
?
1
/ MHz
?
1
/ MHz
g
z
Ni
OGln
?147
H
?
H
?1
H
? H
?2
SO
SO
3
3
-
-
N
N
N
N
Ni
*
N
N
N
N
Ni
n+
HN
H2NOC
H3C

OOC
O
CH3
COO

COO

COO

OOC
O
H
H
O
H2N
Gln
a'147
F430
g
b
a BPS
Br
A
C
D
E
B
181
spectroscopy provides more detailed information on their electronic and spatial structure than
electrical or optical measurements. Furthermore, species with unpaired electrons are often
observed as reaction intermediates, in particular in one-electron transfer processes. Such
processes are the basis of energetics of living cells and occur in many redox reactions. Short-
lived intermediates can be studied with special transient EPR experiments (photosynthetic
reaction centers and light-sensitive proteins) or after spin trapping (Section 10.5.5). For
instance, neurodegeneration during Alzheimers disease is related to an inflammatory process
that is in turn characterized by abnormally high levels of free radicals. EPR spectra recorded in
the presence of the spin trap N-tert-butyl-o-phenylnitrone (PBN) showed that toxic forms of
amyloid-| peptide lead to a formation of radicals. Such radical formation is not observed in the
absence of these peptides or in the presence of a peptide that is rendered nontoxic by
replacement of the methionine residue at position 35 by norleucine (Figure 10.2). This
established the role of residue Met35 in the neurodegenerative process, the probable
involvement of the sulfur atom of this residue in the chemistry of radical formation, and a
potential protective effect of spin traps against Alzheimers disease. Meanwhile PBN and
similar compounds are discussed as potential drugs.
Figure 10.2: Demonstration of radical formation by amyloid-| peptide using spin trap EPR. A) Spin
trap N-tert-butyl-o-phenylnitrone (PBN). B-E) EPR spectra for B) a control solution without
A|-peptide, C) with A| 1-42 added, D) as before, but with residue Met35 replaced by a
norleucine residue, E) with A| 25-35 added. Residues 1-24 and 36-42 appear to be not
essential for radical formation (from: S. Varadarajan et al., J. Struct. Biol. 2000, 130, 184-208.
For such applications EPR is better suited than NMR, mainly because of the low
concentrations involved and the higher sensitivity of EPR. Unless paramagnetic centers relax
very fast, they also cause strong broadening of NMR lines of nuclei in their vicinity. Active
centers of paramagnetic metalloproteins thus cannot be characterized by NMR. On the other
hand, the structure of domains remote from the active center are not accessible to EPR, but can
be studied by NMR. The two spectroscopies are thus often complementary.
B
C
D
E
A
182

Figure 10.3: Structural model of the dimer of the Na
+
/H
+
antiporter of Escherichia coli determined
from the x-ray structure of the monomer by EPR distance measurements between spin-labeled residues.
A) A selected residue is mutated to a cystein and the methanethiosulfonate spin label (MTSSL) is
attached. B) For a homodimer with a C
2
symmetry axis, four degrees of freedom (x, y, u, o) determine
the relative position of the two moieties. C) Nine distances were measured between spin labels in the
two moieties of the dimer were measured (red lines). The dashed line marks a distance that was too
long for the applied technique. The eight known distances overdetermine the structure. D) Interaction
between |-sheets in the two moieties contributes most to binding in the dimer. E) Another contribution
to binding results from contacts between two helices (original publication: Hilger D. et al, Biophys. J.
2007, 93, 3675-3683).
EPR spectroscopy can also complement NMR spectroscopy on diamagnetic systems by
providing access to larger distances between sites and to dynamics on shorter time scales. For
this purpose, stable free radicals have to be introduced in the system. EPR is then used as a
probe technique. Usually, nitroxides (Section 10.5.1) are introduced as non-covalently
attached spin probes or covalently bound spin labels. For proteins site-directed spin labeling
(SDSL) have been developed. This technique is based on mutation of an amino acid near the
site of interest to a cystein and covalent attachment of a thiol-specific spin marker. As is the
case with NMR, structure determination of biomacromolecules by EPR does not require
crystallization.
For instance, the dimer of the Na
+
/H
+
antiporter NhaA of Escherichia coli is weakly
bound and does not survive crystallization. A structural model could be determined from the x-
ray structure of the monomer and measurements of nine distances between spin-labeled
residues in the two moieties of the dimer (Figure 10.3). If the two monomers in a homodimer
are considered as rigid bodies, their relative position is fixed by only four degrees of freedom.
Hence, the problem is overdetermined. The structural model shows that, to a large extent,
binding between the monomers is due to interactions of two |-sheets and to a lesser extent by
N O
S
S
O
O
N O
S
MTSSL
C -SH
b
C -S
b
C
2
x
y
180
f
q
Z
X
Y
A B C
D E
R49
Q47
VII
A
IX
B
R204
V254
L210
W258
183
hydrophobic interactions between two helices. The |-sheet binding motif is common for
soluble proteins, but was found here for the first time for a membrane protein. Later
biochemical experiments confirmed the importance of hydrogen-bonding residues in the |-
sheets for dimer formation.
10.2 Differences between EPR and NMR spectroscopy
The basics of the description of spin systems and spin dynamics are the same for EPR
and NMR. Many of the experimental concepts from NMR can also be applied. In the
remaining lectures we mostly discuss concepts that are particular to EPR. Such concepts result
from the following differences. First, the larger magnetic moment implies frequencies in the
mw range (Table 10.1) that pose more difficult problems for spectrometer technology. Larger
excitation bandwidths are required, which implies pulses with lengths in the nanosecond rather
than microsecond range. For these reasons pulse techniques were developed later in EPR than
in NMR. The typical number of pulses in an experiment is smaller in EPR and pulse shape,
phase, and frequency cannot be varied so easily. Experimental schemes are thus less complex
in EPR compared to NMR.
Due to the larger magnetic moment of the electron spin, typical hyperfine couplings
between an electron and a nuclear spin are by at least two orders of magnitude larger than
typical couplings between two nuclear spins. At the same time, nuclear Zeeman frequencies
are smaller than in NMR as external magnetic fields are usually smaller. Thus, hyperfine
couplings are often of the same order of magnitude as the nuclear Zeeman interaction and lead
to mixing of nuclear spin states. The usual spectroscopic selection rules do no longer apply.
Formally forbidden transitions can be excited by a single pulse or even by continuous
irradiation. This allows for detection of nuclear frequencies in electron spin echo envelope
modulation (ESEEM) experiments (Section 10.4.4). The pulse sequences consist of only mw
pulses that are far off-resonant for the nuclear spins. Such experiments have no equivalent in
NMR. Generally, the spin dynamics is more complex in EPR compared to NMR.
In addition, the larger ratio between couplings and resonance frequencies than in NMR
makes relaxation rates faster compared to the resonance frequencies. As line widths are
proportional to the transversal relaxation rate, this leads to a larger ratio between line widths
184

and resonance frequencies, i.e. to lower resolution. To some extent this lower resolution is
compensated by a larger spread of magnetic parameters. The equivalent to the chemical shift in
NMR is the relative deviation (g-g
e
)/g
e
of the electron g value from the g
e
value for the free
electron. This deviation is typically three orders of magnitude larger than chemical shifts (ppt
instead of ppm).
Because of the larger frequeny shifts and the larger couplings, EPR spectra extend over
a broader frequency range relative to the mean resonance frequency. This requires a larger
relative excitation bandwidth. However, the relative excitation bandwidths of pulses in the
radio frequency (rf) and mw range are almost the same, if the same pulse power is available. It
is thus not usually possible in EPR spectroscopy to excite the whole spectrum by a single
pulse. This eliminates the sensitivity advantage of pulse Fourier transform experiments, so that
continuous-wave (cw) EPR is still the method of choice for recording basic EPR spectra
(Section 10.7.1). Furthermore, cw techniques are applicable even if the transverse relaxation
time T
2
is shorter than the shortest available pulses. Therefore, cw EPR is applicable in a
broader temperature range than pulsed EPR.
NMR spectroscopy relies strongly on the concept of separation of interactions by
multipulse sequences and 2D spectroscopy. These concepts can be transferred to pulse EPR.
By using them, resolution can be improved tremendously and more information can be
obtained. Therefore both cw and pulse EPR are commonly applied. Even in pulsed EPR the
external magnetic field is varied to overcome the limitations in excitation bandwidth.
Another difference arises from the much larger change of the EPR resonance frequency
associated with a change of the orientation of the molecule in the magnetic field. Anisotropies
of EPR frequencies are on the order of tens of Megahertz to several Gigahertz. This matches
the range of inverse rotational correlations times of molecules in fluid solution or soft matter.
Hence, orientations of the molecule exchange on the time scale of cw EPR experiments. The
effect on the spectral line shape can be treated in the same way as chemical-exchange
phenomena in NMR were treated in Chapter 3. The rotational correlation time of the molecule
can then be inferered from the line shape (Section 10.5.2).
Like NMR, EPR spectroscopy can be applied to fluid or solid samples. As the coupling
between two electrons at the same distance is by a factor (u
e
/u
H
)
2
= 436000 larger than
185
between two protons, the concentration of paramagnetic centers needs to be smaller to avoid
excessive line broadening by spin-spin interaction. Best resolution and sensitivity for liquid
samples is achieved at concentrations of of about 10
-3
-10
-5
M. In crystalline samples the
paramagnetic centers are often diluted by doping the paramagnetic compound into an
isomorphous diamagnetic compound. Typical dilutions are 1:100 to 1:10000. If isomorphous
diamagnetic substitution is impossible, solid-state measurements are performed in frozen
solution. Aggregation of the dissolved paramagnetic compound on freezing is avoided by
using glass-forming solvents. A selection of pure glas-forming solvents is given in Table 10.2.
For amphiphilic compounds mixtures of polar and apolar solvents have to be used. Such glass-
forming solvents are, for instance, isopentane/isopropanol in a 8/2 mixture, toluene/acetone in
a 1/1 mixture or with an excess of toluene, and toluene/methanol in a 1/1 mixture or with an
excess of toluene. For biomacromolecules, water/glycerol mixtures are often used.
Table 10.2: Glass-forming solvents with their melting temperatures T
m
and
glass transition temperatures T
g
.
Solvent T
m
/K T
g
/K
2-methylbutane 113.3 68.2
2-methyltetrahydrofuran 137 91
ethanol 155.7 97.2
methanol 175.2 102.6
1-propanol 146.6 109
toluene 178 117.2
ethylene glycol 255.6 154.2
di-1,2-n-butyl phthalate 238 179
glycerol 291.2 190.9
o-terphenyl 329.3 246
poly(styrene) 513 373
186

Another important difference between NMR and EPR is the appearance of the spectra.
For technical reasons (see Section 10.7.1), best sensitivity and resolution in EPR is achieved
with cw experiments at fixed mw frequency. The magnetic field is swept and at the same time
modulated with a typical frequency of 100 kHz. Phase-sensitive detection of the signal
modulation at this frequency yields the derivative of the absorption spectrum (Fig. 10.4).
Although the shape of a derivative Lorentzian absorption line looks like the shape of a
dispersion line, the derivative absorption line has a smaller linewidth.
Figure 10.4: Detection of the derivative of the absorption spectrum by field modulation with
modulation amplitude AB
0
. The amplitude of the reflected mw AV is measured (A) providing a
derivative lineshape (B). To avoid artificial line broadening the condition has to be
fulfilled. The peak-to-peak linewidth AB
pp
= I in field-swept spectra scales as 1/g (roughly with B
0
) if
the transversal relaxation time T
2
does not depend on g (C).
10.3 Interactions and EPR Hamiltonians
10.3.1 Classification of electron spin systems
To avoid excessive relaxational line broadening, EPR spectra are usually measured in a
concentration regime where interactions between paramagnetic centers are negligible. An
exception are systems where the distance between two (or more) paramagnetic centers is
relatively well defined and is measured by EPR (Section 10.3.6). An isolated paramagnetic
center can contain a single unpaired electron (electron spin S=1/2, Section 10.3.3) or several
tightly coupled electron spins (group spin S>1/2, Section 10.3.4). The latter situation is
DB
0
DB
pp
DV
DV
A
B
C
=
2 h
T
2
g
B
3
G
.
B
0
AB
0
3AB
pp
s
187
encountered in high-spin states of transition metal complexes or triplet states of organic
molecules. If the zero-field splitting in such a high-spin system exceeds the mw frequency,
transitions can be induced only with pairs of levels with magnetic quantum numbers of
the electron group spin S. Such a pair of levels is a Kramers doublet, meaning that the levels
are degenerate in the absence of an external magnetic field and are split by the field. The
Kramers doublet can be treated as an effective spin S=1/2 (Section 10.3.5).
Figure 10.5: Topology of spin systems. A) System consisting of seven nuclear spins. All magnetic
moments and hence all 21 spin-spin couplings are comparable. B) System consisting of one electron
spin with a large magnetic moment and six nuclear spins with much smaller moments. Only the six
hyperfine couplings between the electron spin and the nuclear spins are significant.
In most paramagnetic centers the electron spin is coupled to nuclear spins. Because of
the presence of one spin that has a much higher magnetic moment than all the other spins, such
electron-nuclear spin systems have a different topology compared to the nuclear spin systems
observed in NMR. In typical nuclear spin systems all spins exhibit significant couplings to
several other spins (Fig. 10.5A), while in typical electron-nuclear spin systems the nuclear-
nuclear couplings are negligible. Hence, the electron spin couples to many nuclei, but each
nucleus couples significantly only to the one electron spin (Fig. 10.5B). The spectrum of the
nuclear spins is thus easier to analyze and better resolved. It can be observed by ENDOR
(Section 10.4.3) or ESEEM (Section 10.4.4) experiments. Furthermore, spin evolution can be
described by considering only subsystems consisting of the electron spin and one nuclear spin,
since the matrix representation of the spin Hamiltonian factorizes into Hamiltonians of two-
spin systems. This is an important simplification, since two-body problems can be solved
exactly, while many-body problems cannot.
m
S

A B
188

10.3.2 Units of magnetic parameters in EPR spectroscopy
In NMR spectroscopy magnetic parameters are generally given in frequency units or
are dimensionless. In EPR spectroscopy, magnetic field units are also used. Furthermore,
wavenumbers are sometimes given for zero-field splittings and large hyperfine couplings that
may be resolved in optical spectra.
Frequency units and wavenumbers are energy units that can be interconverted without
further knowledge (1 MHz = 3.33564110
-5
cm
-1
). In contrast, the conversion between
magnetic field units and frequencies is not trivial, as it depends on the g value, and the g value
often cannot directly be read off the spectrum. The conversion may thus require a full analysis
of the spectrum. If the g value is known with sufficient precision, we have
. [10.1]
In particular, for (organic radicals), 1 MHz corresponds to 0.0356828 mT or 1
mT to about 28 MHz. The cgs unit Gauss (G) is often used to specify the magnetic field. In fact
it is a unit of magnetic induction (SI unit Tesla (T)), with 10000 G = 1 T. In both NMR and
EPR the magnetic induction is usually referred to as the magnetic field. More precisely, the
magnetic field unit is A m
-1
in SI units or Oersted in cgs units (1 A m
-1
= 4t10
-3
Oersted). If
the relative permeability of a material is unity, the magnetic induction in Gauss is the same as
the magnetic field in Oersted. This is a good approximation for diamagnetic and paramagnetic
substances, but not for ferromagnetic materials. Finally, when discussing spin Hamiltonians,
we use angular frequency units for energies. Angular frequencies e are related to frequencies v
by e=2tv, and to energies E by .
10.3.3 One electron spin S=1/2 coupled to nuclear spins
The spin Hamiltonian in the absence of mw or rf irradiation (static Hamiltonian) is
given by
, [10.2]
AB
0
Avh gu
B
( ) =
g g
e
~
e E h =
H

0 H

EZ H

HF H

NZ H

NQ + + + =
189
where the terms are the electron Zeeman interaction , the hyperfine interaction , the
nuclear Zeeman interaction , and the nuclear quadrupole interaction . In the
following they are discussed in turn.
10.3.3.1 The electron Zeeman interaction
This interaction is formally equivalent to the (nuclear) Zeeman interaction treated in
Sections 5.1 and 5.4. It is given by
, [10.3]
where JT
-1
is the Bohr magneton,
Js Plancks quantum of action, the transpose (T)
of the magnetic field vector , the g tensor, and the electron spin
vector operator . In the PAS of the g tensor, the magnetic field vector can be
written as
, [10.4]
where u and o are polar angles that characterize the direction of this vector in the PAS.
Expansion of Eq. [10.3] in this coordinate system yields
. [10.5]
For an anisotropic g tensor,
1
the direction of the effective field depends on the orientation of
the molecule in the magnetic field. In other words, the quantization axis of the electron spin is
not necessarily parallel to the magnetic field and can have different directions for different
orientations of the molecule. The effective g value is thus given by
. [10.6]
1
Strictly speaking, does not have the transformation properties of a tensor, as it connects two independent
spaces (spin space and laboratory space). Therefore it is sometimes called an interaction matrix. In this lec-
ture, we call all interaction matrices tensors, as most magnetic resonance literature does.
H

EZ H

HF
H

NZ H

NQ
H

EZ
u
B
h
------B0
T
gS

=
u
B
|
e
9.27400899 37 ( )
24
10 = =
h
h 2t ( ) 1.05457159682
34
10 = = B0
T
B0 B
0x
B
0y
B
0z
. . ( ) = g S

x S

y S

z . . ( )
B0 B
0
u o u o u cos . sin sin . cos sin ( ) =
H

EZ
u
B
h
------B
0
g
x
u oS

x g
y
u oS

y sin sin g
z
uS

z cos + + cos sin ( ) =

g
g
eff
g
x
2
u sin
2
o cos
2
g
y
2
u sin
2
o sin
2
g
z
2
u cos
2
+ + =
190

In the high-field approximation, where the electron Zeeman interaction dominates over all the
other interactions, its contribution to the energy of the spin states is proportional to ,
where m
S
is the magnetic quantum number of the electron spin. The resonance field at this
orientation of the molecule with respect to the magnetic field is then
. [10.7]
The deviation of the g value of bound electrons from the g
e
value of the free electron is
mostly due to spin-orbit coupling. As orbital angular momentum is quenched for non-
degenerate ground states, spin-orbit coupling is due to admixture of excited states to the
ground state by the orbital angluar momentum operator . This admixture is usually small and
can thus be treated by perturbation theory. To second order the g tensor is given by
, [10.8]
where indices i and j run over the Cartesian directions x, y, and z and is the Kronecker
delta. The outer sum with index k runs over all atoms in the molecules, where is the spin-
orbit coupling constant for the k
th
atom and and are Cartesian components of the orbital
angular momentum operator of this atom. As spin-orbit coupling is a relativistic effect,
increases with the mass of the atomic nucleus. The inner sum with index m runs over the
molecular orbitals with index 0 designating the singly occupied molecular orbital (SOMO),
where the unpaired electron resides in the ground state. Note that this index can be both
negative (occupied orbitals) and positive (empty orbitals). Accordingly, the energy difference
in the denominator can also be positive or negative. Spin-orbit coupling with empty
p-, d-, or f-orbitals thus leads to negative deviations of g from g
e
(high-field shifts), while spin-
orbit coupling with occupied orbitals leads to positive deviations (low-field shifts). The latter
case is more often encountered. Spin density in s orbitals does not contribute to deviations of g
from g
e
.
The g tensor is thus a complicated function of the frontier orbitals of the molecule. It is
used as fingerprint information for the class of paramagnetic centers. Since light elements (first
and second period) have a small spin-orbit coupling constant, deviations of g from g
e
are only
m
S
g
eff
B
0 res .
hv
mw
g
eff
u
B
--------------- =
l

g
ij
g
e
c
ij
2
k
m l

ki
0 ( , 0 l

kj
m ( ,
E
0
E
m

----------------------------------------------
m 0 =

+ g
e
c
ij
2
k
A
k
( )
ij
k

+ = =
c
ij

k
l

ki
l

kj

k
E
0
E
m

191
of the order of for organic radicals, unless there exist near degenerate frontier
orbitals ( ). Typical cases of orbital degeneracy are the hydroxyl radical OH,
alkoxy radicals RO, and thiyl radicals RS in the gas phase. In condensed phase, degeneracy is
lifted by interaction with neighboring molecules. For these radicals, the g tensor thus strongly
depends on intermolecular interactions. For first row transition metal ions deviations of g from
g
e
are of the order of . They are dominated by contributions from orbitals on the
transition metal ion, so that we find
, [10.9]
where is the spin-orbit coupling constant of the transition metal.
10.3.3.2 The hyperfine interaction
More detailed information on the electronic and spatial structure of the paramagnetic
center can be obtained from the hyperfine couplings. The hyperfine interaction term of the spin
Hamiltonian is given by
, [10.10]
where k runs over all magnetic nuclei (I
k
>0) in the molecule that have hyperfine
couplings larger than the linewidths in the spectrum under discussion. The are nuclear spin
vector operators for these nuclei and the are hyperfine tensors. When analyzing liquid-state
EPR spectra, hyperfine couplings exceeding 3 MHz have to be considered. In solid-state EPR
spectra, such couplings are usually resolved only when they exceed 15 MHz. When analyzing
ENDOR, ESEEM, or hyperfine sublevel correlation (HYSCORE) spectra the resolution limit
is at 0.2-0.5 MHz. Within the high-field approximation the contribution of the hyperfine
coupling to the energies of the spin states is , where A
eff
is an effective hyperfine
coupling.
Hyperfine coupling of the electron spin to a nuclear spin comes about by through-space
dipole-dipole coupling of the two magnetic moments and by the Fermi contact interaction.
The Fermi contact interaction is due to the non-zero probability to find the electron spin at the
10
3
.10
2
E
0
E
m
0 ~
10
2
.10
1
g g
e
1 2
m l

ki
0 ( , 0 l

kj
m ( ,
E
0
E
m

----------------------------------------------
m 0 =

+ g
e
1 2A + = =
H

HF S

T
A
k
I

k
k

=
I

k
A
k
m
S
m
I
A
eff
192

same point in space as the nuclear spin. This happens only when the unpaired electron is in an
s orbital. The Fermi contact interaction leads to a purely isotropic coupling with
, [10.11]
where p
s
is the spin density in the s orbital under consideration, g
n
is the nuclear g value and
JT
-1
the nuclear magneton ( ). The factor
is the probability to find the electron at this nucleus in the ground state with
wavefunction .
1
The magnetic moment of the electron is fully characterized by the g
e
value
of the free electron, as there is no orbital angular momentum in s orbitals.
Unpaired electrons in p-, d, and f- orbitals do not contribute to Fermi contact
interaction, as these orbitals have a node at the nucleus. However, due to the non-spherical
symmetry of these orbitals, dipole-dipole coupling between the magnetic moments of the
electron and nuclear spin does not average. Spin density in such orbitals thus gives rise to
purely anisotropic couplings.
2
In general, the matrix elements T
ij
of the total anisotropic
hyperfine coupling tensor of a given nucleus are computed from the ground state
wavefunction by
. [10.12]
Quantum chemistry programs such as ORCA, ADF, or Gaussian can compute as well as
a
iso
.
A special situation applies to protons, alkali metals and earth alkaline metals, which
have no significant spin densities in p-, d-, or f-orbitals. In this case, the anisotropic
contribution can only arise from through-space dipole-dipole coupling to other centers of spin
density. In a point-dipole approximation the hyperfine tensor is then given by
1
Tabulated in: J. R. Morton, K. F. Preston, J. Magn. Reson. 1978, 30, 577.
2
For given spin densities these couplings can be computed from parameters given in: J. R. Morton, K. F.
Preston, J. Magn. Reson. 1978, 30, 577.
a
iso
S

T
I

a
iso
p
s
2
3
---
u
0
h
-----g
e
u
B
g
n
u
n
v
0
0 ( )
2
=
u
n
|
n
5.05078317 20 ( )
27
10 = = g
n
u
n

n
h =
v
0
0 ( )
2
v
0
T
T
ij
u
0
4th
----------g
e
u
B
g
n
u
n
v
0
( '
3r
i
r
j
c
ij
r
2

r
5
--------------------------- v
0
' , =
T
193
, [10.13]
where the sum runs over all centers with significant spin density p
j
(summed over all orbitals at
this center), the R
j
are distances between the nucleus under consideration and the centers of
spin density, and the are unit vectors along the direction from the considered nucleus to the
center of spin density. For protons in transition metal complexes it is often a good
approximation to consider spin density only at the central ion. The distance R from the proton
to the central ion can then be directly inferred from the anisotropy of the hyperfine coupling.
Admixed orbital angular momentum also contributes to dipole-dipole coupling. This
can be considered by a simple correction, giving the dipole-dipole hyperfine tensor
. [10.14]
We thus obtain for the total hyperfine tensor in Eq. [10.10]
. [10.15]
Note that the product may have an isotropic part, although is purely anisotropic. This
pseudocontact contribution depends on the relative orientation of the g tensor and the spin-
only dipole-dipole hyperfine tensor .
The hyperfine couplings can be used to map the SOMO in terms of a linear
combination of atomic orbitals. For catalytic species this can provide insight into reactivity and
thus into the mechanism of catalysis. For organic compounds that form reasonably stable
radical anions or cations the SOMO of the radical anion corresponds to the LUMO of the
parent compound and the SOMO of the radical cation to the HOMO of the parent compound.
The contributions discussed so far can be understood in a single-electron
representation. A further contribution arises from correlation between electrons in the
molecule. Assume that the p
z
orbital on a carbon atom contributes to the SOMO, so that the o
spin state of the electron is preferred in that orbital (Fig. 10.6). Electrons in other orbitals on
the same atom will then also have a slight preference for the o state, as electrons with the same
T
k
u
0
4th
----------g
e
u
B
g
n
u
n
p
j
3n
j
n
j
T
1
R
j
3
-----------------------
j k =

=
n
j
A
dd
gT
g
e
------- =
A
k
A
k
a
iso k .
1
gT
k
g
e
--------- + =
gT
k
T
k
T
k
194

spin tend to avoid each other and thus have less electrostatic repulsion.
1
In particular, this
means that the spin configuration in Fig. 10.6A is slightly more preferable than the one in Fig.
10.6B. According to the Pauli principle the two electrons that share the o bond orbital of the C-
H bond must have antiparallel spin. Thus, the electron in the s orbital of the hydrogen atom
that is bound to the spin-carrying carbon atom has a slight preference for the | state. This
corresponds to a negative isotropic hyperfine coupling of the directly bound o proton, which is
induced by the positive hyperfine coupling of the adjacent carbon atom. The effect is termed
spin polarization, although it is entirely different from the polarization of electron spin
transitions in an external magnetic field.
Figure 10.6: Spin polarization on an adjacent hydrogen atom (o proton) induced by spin density in a p
z
orbital on carbon. The spin configuration in A) is slightly favored with respect to the one in B).
Figure 10.7: Isotropic hyperfine coupling of a next-neighbor hydrogen atom (| proton) induced by
hyperconjugation. The overlap of the carbon p
z
orbital and the hydrogen s orbital depends on dihedral
angle ;.
Spin polarization is observed both in o radicals, where the spin density is confined to a
single p
z
orbital, and in t radicals, where the spin density is delocalized in a t system. In t
1
This preference for electrons on the same atom to have parallel spin is also the basis of
Hunds rule.
C C H H
s s
p
z
p
z
A B
C
H
H
c
A B
195
radicals spin polarization is the only contribution to the isotropic hyperfine coupling of a
proton that is directly bound to a carbon atom of the conjugated system. The isotropic
hyperfine coupling of such an o proton can thus be predicted by the McConnell equation
, [10.16]
where p
t
is the spin density at the adjacent carbon atom and Q
H
is a parameter of the order of
mT, which slightly depends on the structure of the t system.
The isotropic hyperfine coupling of | protons in o radicals is caused by spin
delocalization from the spin-carrying p
z
orbital to the s orbitals on the | hydrogen atoms.
These orbitals are sufficiently close in space to allow for overlap. The extent of this
hyperconjugation depends on the dihedral angle between the p
z
orbital lobes and the C-H
|
bond. When the C
|
group rotates freely with respect to the spin-carrying orbital, an average
hyperfine coupling is observed. Unlike spin polarization, hyperconjugation does not depend on
electron correlation, but can rather be seen as spin density transfer.
10.3.3.3 Nuclear Zeeman interaction
The nuclear Zeeman interaction can be neglected in the analysis of EPR spectra, unless
it is of the same order of magnitude as a resolved hyperfine coupling of the same nucleus. In
analysis of ENDOR, ESEEM, and HYSCORE spectra, the nuclear Zeeman interaction is used
to assign hyperfine couplings to elements (isotopes). This information is missing in EPR
spectra.
Chemical shift information cannot be obtained from EPR, ENDOR, ESEEM, or
HYSCORE spectra, as chemical shifts are smaller than the linewidths for nuclear spins with
significant hyperfine couplings. Chemical shift is thus neglected in the nuclear Zeeman
Hamiltonian, which is given by
. [10.17]
In the laboratory frame this simplifies to
, [10.18]
A
iso H .
Q
H
p
t
=
2.5
H

NZ
g
n
u
n
h
-----------B0
T
I

=
H

NZ e
I
I
z
=
196

with e
I
= -
n
B
0
. Within the high-field approximation the nuclear Zeeman interaction
contributes an energy m
I
e
I
to the spin states.
At S-band to Q-band frequencies the nuclear Zeeman frequency is often comparable to
hyperfine couplings or smaller. This leads to state mixing by anisotropic hyperfine
interactions, which is the basis of ESEEM and HYSCORE experiments. At W-band
frequencies and above this phenomenon is observed only for a few low- nuclei with large
hyperfine couplings (large spin densities), such as for nitrogen atoms that are directly
coordinated to a transition metal ion.
10.3.3.4 Nuclear quadrupole interaction
The nuclear quadrupole interaction (Section 5.9) does not contribute to EPR spectra
unless it is of the same order of magnitude as the hyperfine coupling of the same nucleus and
the hyperfine coupling of this nucleus is resolved. Usually this happens only for heavy
elements from the third row of the periodic table onwards. In analyzing ENDOR, ESEEM, and
HYSCORE spectra of nuclei with spin I
k
>1/2 the nuclear quadrupole interaction has to be
considered. In an EPR context, the nuclear quadrupole Hamiltonian is often written as
, [10.19]
where the principal values of the nuclear quadrupole tensor are given by
, [10.20]
, [10.21]
and
. [10.22]
The principal values are thus directly related to the field gradient components V
xx
, V
yy
, and V
zz
defined in Section 5.9. Within the high-field approximation the contribution of the nuclear
quadrupole interactions to the energies of the spin states is proportional to , where P
eff
is an effective nuclear quadrupole interaction. To first order, the transition energy for a pair of
H

NQ I

T
PI

=
P
z
e
2
qQ
2I 2I 1 ( )h
---------------------------- =
P
x
P
y
nP
z
=
P
x
P
y
P
z
+ + 0 =
m
I
2
P
eff
197
levels thus does not depend on the nuclear quadrupole interaction. In particular, this
applies to the allowed transition .
10.3.4 One electron group spin S>1/2 coupled to nuclear spins
If m electrons are distributed indegenerate orbitals, each orbital is first singly occupied
(Fig. 10.8A, Hunds rule). This is because electron pairing in the same orbital would lead to
increased Coulomb repulsion. In the presence of a ligand field that removes orbital
degeneracy, pairing is preferred if the energy difference between the upper and lower set of
orbitals exceeds electron pairing energy (Fig. 10.8B,C). Depending on the strength of the
ligand field, several electrons may thus be unpaired (high-spin state) or paired as far as
possible (low-spin state).
Figure 10.8: High-spin and low-spin configurations for a d
5
transition ion, such as Fe
3+
. A) In the
absnece of a ligand field all unpaired electrons in the degenerate d orbitals have parallel spin (high spin
S= 5/2). B) In a weak ligand field, where the splitting between the e
g
and t
2g
levels is smaller than the
electron pairing energy, all spins are parallel (high spin, S= 5/2). C) In a strong ligand field, where the
level splitting exceeds the electron pairing energy, all electrons occupy the set of lowest-lying orbitals,
where they pair (low spin, S= 1/2).
In high spin states of transition metal ions all the unpaired electrons reside mainly in
orbitals on the same atom. They are so close in space that they couple strongly and cannot be
excited separately from each other. Hence, it is appropriate to describe them as a single group
spin S = m/2. For instance, Fe
3+
with a 3d
5
configuration in weak ligand fields assumes an S =
5/2 high-spin state, while in strong ligand fields it assumes an S = 1/2 low-spin state.
On first sight one might expect that the distribution of five unpaired electrons on all
five 3d orbitals is spherically symmetric so that no equivalent to the nuclear quadrupole
m
I

m
I
1 2 1 2 ( ) =
A B C
d
e
g
t
2g
t
2g
e
g
198

interaction would result. However, spin-orbit coupling breaks this symmetry. Electron-group
spins S > 1/2 thus have an additional interaction that is formally analogous to the nuclear
quadrupole interaction. This zero-field splitting
1
contribution to the spin Hamiltonian is
written as
, [10.23]
where is the traceless zero-field splitting tensor. In the principal axes frame of this tensor,
the Hamiltonian can be written as
. [10.24]
with D = 3D
z
/2 and E = (D
x
-D
y
)/2. By convention, D
z
is the principal value with the largest
magnitude, so that E cannot exceed D/3. For axial symmetry, E = 0. In cubic symmetry,
quadrupolar zero-field splitting vanishes (D= E = 0). In that case and for S > 3/2, the much
smaller hexadecapolar contribution may become observable.
For transition metal ions, g shifts with respect to g
e
(Eq. [10.9]) and zero-field splitting
are related,
. [10.25]
Within the high-field approximation the contribution of the zero-field splitting to the energy
levels is proportional to . Thus the transition that is allowed in
EPR spectra of half-integer group spins S is not affected to first order by zero-field splitting.
10.3.5 Effective spin S=1/2 in a high-spin system S>1/2
If the zero-field splitting is much larger than the mw quantum and the electron Zeeman
interaction at accessible magnetic fields, only part of the transitions of a spin S > 1/2 are
accessible for EPR. In conventional EPR at X band, the limit is at about 20 GHz. For integer
group spins S, none of the transitions is accessible unless the zero-field splitting tensor has at
1
The term of the Hamiltonian is sometimes called fine structure term instead of zero-field splitting.
H

ZF S

T
DS

=
D
H

ZF
PAS ( )
D
x
S

x
2
D
y
S

y
2
D
z
S

z
2
+ + =
D S

z
2
1
3
---S S 1 + ( ) E S

x
2
S

y
2
( ) + =
D
2
A =
m
S
2
D
eff
m
S
1 2 1 2 ( ) =
199
least axial symmetry. Such integer-spin systems are termed EPR silent, although they are
paramagnetic and can be studied at higher fields and frequencies. For half-integer group spins
there is at least one pair of low-lying levels that are degenerate in the absence of a magnetic
field and are split in its presence. Such a Kramers doublet can be described as an effective spin
S = 1/2. For instance, for high-spin Fe
3+
(S = 5/2) in non-cubic symmetry, EPR transitions can
be observed only within the three Kramers doublets, but not between them (Fig. 10.9). The
spin Hamiltonian for each Kramers doublet is equivalent to the spin Hamiltonian of a spin S =
1/2 (Section 10.3.3), except that the effective g values depend on the zero-field splitting
parameters D and E and are not strictly field-independent.
Figure 10.9: Kramers doublets with effective spin S=1/2 for high-spin Fe
3+
(3d
5
) with E/D = 1/3. The
mw quantum (vertical bars) is too small to excite transitions between different Kramers doublets. The
three Kramers doublets have different effective g values.
10.3.6 A pair of weakly coupled electron spins
10.3.6.1 Exchange coupling
Consider two electron spins S
1
= 1/2 and S
2
= 1/2 as individual paramagnetic centers. At
distances up to at least 15 there is still significant overlap of the two SOMOs, so that the two
electrons can exchange. This exchange interaction leads to a splitting between the singlet state
(group spin S = 0) and the triplet state (group spin S = 1) of the coupled system. If this splitting
B
0
0
g = 9.67
g = 4.3
g = 0.6
200

is so small that transitions between the singlet and triplet state can be excited in an EPR
experiment, it is more convenient to treat the system in terms of two individual spins that are
coupled to each other. Typically at distances larger than 5-10 the individual spin treatment is
applied.
The energy difference between the singlet and triplet state is the exchange integral
, [10.26]
where v
1
and v
2
are the wavefunctions of the two unpaired electrons.
1
For positive J, the
singlet state is lower in energy, i.e., the orbital overlap is bonding and the interaction is
antiferromagnetic. Negative J correspond to a lower-lying triplet state, i.e., antibonding orbital
overlap and a ferromagnetic interaction.
The exchange term of the spin Hamiltonian is given by
. [10.27]
This term describes a scalar (purely isotropic) coupling. Anisotropic contributions to the
exchange coupling may occur for species involving heavy elements, but can be neglected for
organic radicals. Within the high-field approximation the contribution of the exchange
interaction to energy levels is proportional to m
S,1
m
S,2
J.
To a good approximation the exchange interaction decays exponentially with distance r
between the paramagnetic centers. The decay rate depends on the conductivity of the medium
between the centers and on the presence or absence of a conjugated network of bonds between
them. If conjugation is weak and the matrix isolating, exchange coupling is much smaller than
the dipole-dipole coupling through space at distances longer than 15 .
1
There exist different conventions for the sign of J and the factor 2 may be missing. One should always
ascertain which convention a certain author is adhering to.
J 2e
2
v
1
-
r
1
( )v
2
-
r
2
( )v
1
r
2
( )v
2
r
1
( )
r
1
r
2

------------------------------------------------------------------------ r
1
d r
2
d

=
H

EX JS

1
T
S

2 J S

1xS

2x S

1yS

2y S

1zS

2z + + ( ) = =
201
10.3.6.2 Dipole-dipole coupling
The dipole-dipole coupling between two electron spins is analogous to that between
two nuclear spins (Section 5.8). For transition metal and rare earth ions the g anisotropy is so
large that the two magnetic moments are not parallel to the magnetic field. The interaction
energy according to Eq. [5.74] is then parametrized by three angles u
1
, u
2
, and o (Fig. 10.10)
and is given by
. [10.28]
The dipole-dipole coupling term of the spin Hamiltonian assumes the form
. [10.29]
Figure 10.10: Coupling between two magnetic moments and in a general orientation with
respect to the external magnetic field and definition of the angles u
1
, u
2
, and o that parametrize this
interaction. Note that these angles implicitly depend on the orientation of the molecule in the magnetic
field.
In the special case of two parallel magnetic moments, which is a good approximation
for two organic radicals, the dipole-dipole coupling tensor has the principal values -e
dd
, -
e
dd
, 2e
dd
with
. [10.30]
The orientation of the molecule can then be characterized by a single angle u between the
magnetic field axis and the spin-spin vector. The orientation-dependent dipolar splitting thus
assumes the form
. [10.31]
E
u
0
4t
------ u
1
u
2
1
r
3
---- 2 u
1
u
2
cos cos u
1
u
2
o cos sin sin ( ) =
H

dd S

1
T
DS

2
1
r
3
----
u
0
4th
---------- g
1
g
2
u
B
2
S

1S

2
3
r
2
---- S

1r ( ) S

2r ( ) = =
r
0
B

q
1
q
2
f
u
1
u
2
B0
D
e
dd
1
r
3
----
u
0
4th
---------- g
1
g
2
u
B
2
=
d u ( ) 3 u cos
2
1 ( )e
dd
=
202

For organic radicals, is a good approximation, so that the dipole-dipole
coupling v
dd
=e
dd
/2t in frequency units is given by
. [10.32]
With typical transversal relaxation times T
2
of electron spins of a few microseconds,
dipole-dipole couplings can be measured down to about 100 kHz in favourable cases,
corresponding to distances of 8 nm. For soluble proteins and membrane proteins reconstituted
into detergent micelles this upper distance limit may reduce to about 6 nm and for membrane
proteins reconstituted into liposomes to about 5 nm. These limits are comparable to the
diameter of protein molecules.
Within the high-field approximation the contribution of the dipole-dipole interaction to
energy levels is proportional to m
S,1
m
S,2
e
dd,eff
. This is the same dependence on magnetic
quantum numbers as for the exchange coupling. The two contributions to electron-electron
coupling thus cannot be separated by spin manipulation.
Figure 10.11: Transversal and longitudinal relaxation of the electron spin of a nitroxide molecule by a
collsion with triplet dioxygen. (a) The molecules are separated and diffuse towards each other. For
example, both unpaired electrons of the oxygen molecule are in the | state, while the unpaired electron
of the nitroxide molecule is in the o state. (b) The molceules collide, their orbitals overlap, and the three
unpaired electrons are no longer distinguishable. (c) The molecules have separated again. With a
probability of 2/3 the nitroxide molecule is now left with one of the unpaired electrons that originally
belonged to the oxygen molecule (and vice versa). The electron spin of the nitroxide thus has lost phase
memory and has changed its spin state.
10.4 Measurement of hyperfine couplings
10.4.1 cw and echo-detected EPR spectroscopy
Hyperfine couplings manifest in the spectra of both electron and nuclear spins. Large
hyperfine couplings (>20...50 MHz or 0.7...2 mT at g = g
e
) can often be determined with
g
1
g
2
g
e
~ ~
v
dd
52.04 MHz
r
3
nm
3

----------------------------- =
N N
N
O O
O
O=O O=O O=O
a b c
203
sufficient precision from EPR spectra even in the solid state. For small radicals in solutions
with low viscosity, precision of an EPR measurement may even be sufficient for hyperfine
couplings as small as 3 MHz. In these situations, cw EPR or field-swept echo-detected EPR is
the method of choice, as it is more sensitive and technically easier than measurement of the
nuclear spectrum. To obtain utmost hyperfine resolution, line broadening due to couplings
between electron spins has to be avoided. In the solid state this requires concentrations of 1
mmol L
-1
or less. .
Figure 10.12: Line broadening due to collisonal exchange. 1,8-Dimethyl naphthaline radical anion at a
concentration of a) 10
-3
M, b) 10
-5
M. c) Wursters blue without (top) and with (bottom) oxygen in the
solution.
In the liquid state linewidths are smaller. Furthermore, exchange broadening can also
arise due to collisions between paramagnetic molecules. During such a collision the orbitals
overlap, the unpaired electrons of both molecules become indistinguishable and may be
exchanged when the molecules separate again (Fig. 10.11). This leads to sudden changes in
resonance frequency and thus to phase relaxation. The transversal relaxation time T
2
is
shortened and lines are broadened. In exceptional cases, concentrations down to 200 umol L
-1
may be required to avoid such broadening (Fig. 10.12A).
Exchange broadening is also caused by dissolved oxygen, as dioxygen has a triplet
ground state and is thus paramagnetic. The effect is often tolerable for measurements in
aqueous solution, but highly detrimental for measurements in unpolar solvents, where oxygen
solubility is much higher (Fig. 10.12B).
c)
204

.
Figure 10.13: Simulated EPR spectrum of the phenyl radical (bottom) and schematic drawing of how
the splitting pattern arises (top).
The hyperfine splitting pattern of radicals in solution is generated in the same way as
discussed in Section 7.1 for J-coupled spectra of weakly-coupled nuclear spins in NMR, except
that only the spectrum of one of the spins (the electron spin is observed). Furthermore, the
number of nuclear spins with significant hyperfine coupling to the electron spin is usually
larger than the corresponding number in J-coupled NMR spectra. As a particularly simple case,
the EPR spectrum of the phenyl radical is shown in Fig. 10.13. This radical can be generated
for instance by photolysis of phenylbromide in solution. Due to the low natural abundance of
13
C, carbon hyperfine splittings are apparent only in weak satellite lines that do not concern us
here. The splitting pattern is thus entirely due to proton hyperfine couplings. Among the
protons, the ones in positions 2 and 6 are magnetically equivalent with a coupling of 1.743 mT,
the ones in positions 3 and 5 with a coupling of 0.625 mT and the proton in position 4 has a
2H(1.743 mT)
2H(0.625 mT)
1H(0.204 mT)
375 380 385
B
0
(mT)
H H
H
H
H
2 6
3
5
4
205
coupling of 0.204 mT. The resulting spectrum, a triplet of triplets of doublets has 18 lines. As
the pattern is clearly apparent, the hyperfine couplings can be read off directly.
Figure 10.14: Experimental EPR spectrum of the paracyclophan free radical.
Such a simple analysis may no longer be possible if the subpatterns overlap. This
happens almost with certainty when the number of hyperfine coupled nuclei increases, as the
number of lines in the EPR spectrum increases multiplicatively
, [10.33]
where index i runs over the groups of equivalent nuclei, the k
i
are the numbers of nuclei within
each group, and the I
i
their nuclear spin quantum numbers. For instance, for the free cyclophan
radical in solution with 4 equivalent aromatic protons, 9 equivalent exo, and 9 equivalent endo
protons, lines arise (Fig. 10.14). Coordination with a K
+
ion (nuclear spin I =
3/2 for
39
K, quadruplet splitting) removes the equivalence between the two moieties of the
molecule, so that each group of equivalent protons splits into two inequivalent subgroups. The
number of lines increases to .
Spectra like this are difficult to analyse. Due to the special topology of electron-nuclear
spin systems (Fig. 10.5B), the nuclear spectrum in the liquid state has a much smaller number
of lines
n
EPR
2k
i
I
i
1 + ( )
i
I
=
5 9 9 405 =
3 3 5 5 5 5 4 22500 =
206

, [10.34]
where i again runs over the groups of equivalent nuclei. For instance, the nuclear spectra of
phenyl radicals, uncoordinated paracyclophan radicals, and K
+
-coordinated paracyclophane
radicals have only 6, 6, and 14 lines, respectively. In the solid state, additional splitting due to
nuclear quadrupole couplings leads to a number of lines
. [10.35]
Among our examples, this would change the number of lines only for K
+
-coordinated
paracyclophane radicals to 18.
Figure 10.15: Typical S = 1/2 EPR spectra in the absence of hyperfine couplings. The frequency
dispersion is caused only by a g tensor with axial symmetry (A,B) or orthorhombic symmetry (C,D).
The low-field and high-field edges of the absorption spectra (A,C) correspond to selection of a small
subset of orientations near the x or z axis of the g tensor PAS. The first derivative lineshape, as detected
in cw EPR spectroscopy, is dominated by the singularities that arise at principal axis directions.
n
NMR liq .
2S 1 +
i

=
n
NMR sol .
2I
i
2S 1 + ( )
i

=
300 320 340
h g n m
mw B
/
||
h g n m
mw B
/
z
h g n m
mw B
/
||
h g n m
mw B
/
z
h g n m
mw B
/
x
h g n m
mw B
/
^
h g n m
mw B
/
y
h g n m
mw B
/
^
h g n m
mw B
/
x
h g n m
mw B
/
y
300 320 340
B
0
(mT) B
0
(mT)
q = 0
q = 90
300 320 340
A C
B D
300 320 340
207
The nuclear spin spectra cannot be measured with an NMR spectrometer, as the
hyperfine splittings exceed the bandwidth of NMR probeheads by orders of magnitude.
Furthermore, detection at the low NMR frequency insted of the high EPR frequency would
lead to a drastic loss in sensitivity. For these reasons spectra of hyperfine coupled nuclei are
detected by EPR-based methods such as ENDOR or ESEEM.
The dispersion of electron spin resonance frequencies due to g anisotropy for powders
or frozen solutions causes a substantial drop in hyperfine resolution compared to the liquid
state. Even for transtion metal ions with only moderate g anisotropy, such as V(IV), Cu(II), or
Co(II) the spectrum in the absence of any hyperfine couplings extends over tens of mT (Fig.
10.15).
Except for the singularities corresponding to principal axis directions, the first
derivative of the absorption lineshape is barely detectable. If hyperfine anisotropy is much
smaller than g ansiotropy or if the PAS of the g and hyperfine tensors coincide, observable
hyperfine splittings thus correspond to the principal axis directions of the g tensor. If g and
hyperfine anisotropy are of the same order of magnitude and the PAS are non-coincident,
spectrum analysis requires lineshape simulations.
Figure 10.16: Hyperfine splittings in solid state EPR spectra of powders, glasses or frozen solutions.
(simulations for a square planar copper(II) complex with four equal ligands L). A) Each nuclear spin
state gives rise to a separate axial powder pattern. B) The absorption spectrum, as detected by field-
swept echo-detected EPR is the sum of the four separate patterns. C) The derivative of the absorption
spectrum, as detected by cw EPR, exhibits hyperfine splitting along g
||
. The inset shows a stereoview of
the complex with the unique axis of the g and copper hyperfine tensor.
0.3 0.32 0.34 0.3 0.32 0.34
A
B
C
m
I
= |-3/2
m
I
= |-1/2
m
I
= |+1/2
m
I
= |+3/2
B
0
(T) B
0
(T)
h g n m
mw B
/
||
hfi at g
||
A
||
h g n m
mw B
/
^
hfi at g
^
Cu
L
L L
L
g
||
,A
||
208

A simple case is illustrated in Fig. 10.16 on the example of a square planar copper
complex with four equal ligands L. This species has a C
4
symmetry axis. A C
n
symmetry axis
with implies an axial g tensor with its unique axis coinciding with the symmetry axis. As
the SOMO also has C
4
point symmetry at the Cu
2+
ion, the copper hyperfine tensor is also
axial and has the same unique axis. For each magnetic quantum number m
I
= -3/2, -1/2, +1/2,
and +3/2 the two tensors for the nuclear Zeeman and copper hyperfine interaction add to a total
axial tensor that describes the anisotropy of the resonance frequency.
1
Each nuclear spin state
thus gives rise to a separate axial powder pattern (Fig. 10.16A). The edges of the patterns are
shifted with respect to each other by multiples of the hyperfine coupling A
||
(low-field edge)
and (high-field edge). The small splitting is usually unresolved in the EPR spectrum
(Fig. 10.16B). The parameters g
||
, , and A
||
can be directly read off the cw EPR spectrum
(Fig. 10.16C).
10.4.2 Nuclear spin spectra
Allowed transitions in nuclear spin spectra involve a change of the magnetic quantum
number of the nuclear spin m
I
by unity while the magnetic quantum number of the electron
spin m
S
remains constant, i.e., they are of the type . If the high-field
approximation applies to both the electron and nuclear spin, the angular frequencies of such
transitions are given by
. [10.36]
For simplicity, the following considerations are restricted to a system consisting of a nuclear
spin I=1/2 coupled to an electron spin S=1/2. This system has two nuclear transitions with the
angular frequencies
. [10.37]
Depending on the sign of the gyromagnetic ratio of the nuclear spin, the sign of the hyperfine
coupling, and the relative magnitudes of the two interactions, the transition frequencies can be
1
The argument applies strictly only in frequency domain, but the qualitative conclusions are true also in
field domain.
n 3 >
A

m
I
m
I 1 +

Ae e
I
m
S
A
eff
2m
I
1 + ( )P
eff
+ + =
Ae e
I
A
eff
2
---------- =
209
either negative or positive and they can have either the same or a different sign. The absolute
sign of the frequency can only be detected if the nuclear transitions are directly excited with
circularly polarized radiofrequency (rf) irradiation. As linearly polarized irradiation is used
this information is lost. The relative sign can be detected in experiments that correlate the two
transitions, such as in the hyperfine sublevel correlation (HYSCORE) experiment (Section
10.4.2.3).
The sign uncertainty complicates assignment and interpretation of the spectra. This is
first discussed for the case of isotropic hyperfine coupling. The same considerations apply to
spectra of single crystals.
10.4.2.1 Isotropic case
Consider the case of the phenyl radical whose EPR spectrum is shown in Fig. 10.13. At
X-band frequencies the nuclear Zeeman frequency v
I
=e
I
/2t is about -15 MHz. The hyperfine
couplings are A
1
/2t = 5.7 MHz for the para proton H
4
, A
2
/2t = 17.5 MHz for the meta protons
H
3
and H
5
, and A
3
/2t = 48.8 MHz for the ortho protons H
2
and H
6
. The couplings of the para
and meta protons are in the weak coupling regime
(weak coupling) [10.38]
while the couplings of the ortho protons are in the strong coupling regime
(strong coupling) . [10.39]
In the weak coupling case the doublet is centered at |v
I
| and split by |A|/2t (Fig. 10.17A,B).
1
In
the strong coupling case, one of the frequencies has opposite sign, corresponding to precession
of the magnetization with an opposite sense of rotation. As the sign is lost, the corresponding
line is mirrored to positive frequencies. The doublet is thus centered at |A|/4t (|A|/2 on an
angular frequency scale) and split by 2|v
I
|. When different nuclear isotopes contribute to the
spectrum, transitions of strongly coupled low- nuclei, e.g.
14
N, may thus overlap with
transitions of weakly coupled high- nuclei, e.g.
1
H. This problem is common at X-band
1
In the literature, A is often used as an angular frequency in equations but as a frequency in spectra or
tables. Beware!
A 2 e
I
<
A 2 e
I
>
210

frequencies and below. The overlap may be eliminated by going to higher frequencies or by
HYSCORE.
10.4.2.2 Anisotropic case
In the regime of very weak coupling , the high-field approximation is valid
for the nuclear spins. The nuclear spins are then quantized along the direction of the external
magnetic field. The hyperfine interaction perpendicular to this direction is truncated, and Eq.
[10.37] applies with A
eff
= A
z
being the hyperfine coupling along the external field direction.
Figure 10.17: Schematic nuclear spectrum of the phenyl radical. A) Subspectrum of the para proton
(weak coupling). B) Subspectrum of the meta protons (weak coupling). C) Subspectrum of the ortho
protons (strong coupling). The line at (relative) negative frequencies is mirrored to positive
frequencies. D) Total spectrum.
If the nuclear Zeeman and hyperfine interaction are of the same order of magnitude, the
hyperfine field at the nucleus perpendicular to the external field (terms and
of the Hamiltonian) can no longer be neglected. Instead of being non-secular, as in the high-
field approximation, these terms are now pseudosecular. Together they contribute an effective
interaction in the xy plane of the laboratory frame. The corresponding field
A e
I

w( H)
1
2w( H)
1
w( H)
1
w( H)
1
w( H)
1
0
0
0
0
C
B
A
D
A
3
A
1
A
2
A
3
/2
w
nuclear
w
nuclear
w
nuclear
w
nuclear
A
zx
S

zI

x A
zy
S

zI

y
B A
zx
2
A
zy
2
+ =
211
at the electron spin (terms and of the Hamiltonian) is usually still negligible
as are the remaining off-diagonal terms, since the high-field approximation still applies to the
electron spin with its much larger Zeeman interaction. Thus, the truncated Hamiltonian for the
S=1/2, I=1/2 spin system becomes
. [10.40]
A new x direction was chosen for the nuclear spin frame to simplify the Hamiltonian.
1
The
Hamiltonian is written in a singly rotating frame, where the electron spin space rotates with the
mw frequency while the nuclear spin space is fixed with respect to the laboratory frame.
Hence, O
S
is a resonance offset, while e
I
is the total nuclear Zeeman frequency.
Figure 10.18: Local fields on the nucleus in the m
S
=1/2 (o) and m
S
=-1/2 (|) states. The effective field
directions for the two states differ; they are tilted by angles n
o
and n
|
with respect to the external field
direction z.
When divided by the gyromagnetic ratio
n
, the Hamiltonian terms containing I spin
operators can be interpreted as local fields at the nuclear spin (Fig. 10.12). The pseudosecular
contribution causes a tilt of the effective field away from the external field direction z. As the
pseudosecular contribution has different signs for the o and | states of the electron spin, the
effective field axis ist tilted by angles n
o
and n
|
in different directions. As the effective field
component along z is once the sum and once the difference of the nuclear Zeeman field and the
secular hyperfine field, the magnitudes of n
o
and n
|
also differ. The quantization axis of the
nuclear spin thus is no longer well defined and m
I
is no longer a good quantum number.
1
This is convenient, unless the nuclear spins are directly irradiated by rf.
A
xz
S

xI

z A
yz
S

yI

z
H

0 O
S
S

z e
I
I

z AS

zI

z BS

zI

x + + + =
z
x
A/2
-A/2
- /2 B B/2
w
I
2
h
a
h
h
b
w
w
b
a
a
b
b
a
212

This has two important consequences. First, an mw pulse that excites the electron spin
will also have an influence on nuclear spin state, as the direction of the local field at the
nucleus is changed. To some extent the pulse thus excites forbidden transitions in which both
m
S
and m
I
change. This is the basis of ESEEM experiments (Section 10.4.4). Second, Eq.
[10.37] for the nuclear frequencies does no longer apply. The two nuclear frequencies are now
given by
[10.41]
and
, [10.42]
as can be inferred by vector addition (Fig. 10.18). The doublet is now centered at a frequency
that is slightly higher than e
I
while the splitting is slightly smaller than A.
For the hyperfine and nuclear Zeeman field along z cancel exactly for one
of the two electron spin states, either o or |. In this situation of exact cancellation the effective
field is within the xy plane, the nuclear frequency is B/2, and the transition moments of
allowed and forbidden transitions are nearly the same. For quadrupolar nuclei (I>1/2) with
small hyperfine anisotropy the nucleus at exact cancellation experiences a near zero-field
situation. Narrow lines at the zero-field nuclear quadrupole resonance (NQR) frequencies are
then observed.
In macroscopically disordered systems, such as powders, glasses, and frozen solutions,
A and B depend on the relative orientation of the hyperfine tensor PAS and the magnetic field.
For a hyperfine tensor with axial symmetry and principal values (a
iso
+2T, a
iso
-T, a
iso
-T), the
orientation dependence is fully described by the angle u between the unique axis of the
hyperfine tensor and the magnetic field,
, [10.43]
. [10.44]
e
o
e
I
A
2
---- +
\ .
| |
2
B
2
4
------ + =
e
|
e
I
A
2
----
\ .
| |
2
B
2
4
------ + =
A 2 e
I
=
A a
iso
T 3 u 1 cos
2
( ) + =
B 3T u u cos sin =
213
Powder patterns computed from Eq. [10.41-10.44] are shown for weak coupling, exact
cancellation, and strong coupling in Fig. A, B, and C, respectively.
Figure 10.19: Simulated anisotropic nuclear frequency spectra for an S=1/2, I=1/2 system with weak
coupling (A), at exact cancellation (B), and with strong coupling (C).The same sign is assumed for a
iso
and e
I
.
10.4.2.3 Hyperfine sublevel correlation (HYSCORE)
The frequencies of a pair of transitions of the same nucleus in the electron spin o and |
manifold can be correlated in a 2D experiment by using a mw pulse for mixing. To keep
with usual notation in EPR literature flip angles are given in radians from now on. A
pulse is thus a t pulse and a pulse a t/2 pulse. The correlation peaks created by mixing
with the t pulse appear in the quadrant ( , ) for weakly coupled nuclei, while they
appear in the quadrant ( , ) for strongly coupled nuclei. A schematic spectrum for
the phenyl radical is shown in Fig. 10.20 and can be compared to the EPR spectrum in Fig.
10.13 and the one-dimensional nuclear spectrum in Fig. 10.17. For macroscopically disordered
systems with anisotropic hyperfine coupling, curved ridges result. The anisotropy of the
hyperfine coupling can be extracted from their curvature and their shift with respect to the
antidiagonal at e
I
, even if only part of the correlation ridge can be observed (Fig. 10.21).
A
B
C
w
I
w
I
0
0
0
a
iso
/2
B/2
w
a
w
a
w
b
w
b
180
180
90
e
1
0 > e
2
0 >
e
1
0 > e
2
0 <
214

Figure 10.20: Schematic HYSCORE spectrum for the phenyl radical. The two doublets of the weakly
coupled para and meta protons are separated from the doublet of the strongly coupled ortho protons.
Figure 10.21: Schematic HYSCORE spectrum for a macroscopically disordered system with
ansiotropic hyperfine coupling.The dashed line is the e
I
antidiagonal.
n( H)
1
-n( H)
1
0
n
2
n
2
2
n
(
H
)
1
A
3
/2
A
1
A
2
2
+
T
a
i
s
o
T
a
+
i
s
o
9
T 3
2
I
I
w
I
2
w
I
a
n
t
i
d
i
a
g
o
n
a
l
215
10.4.3 ENDOR spectroscopy
10.4.3.1 Davies ENDOR
The polarization of electron spin transitions is at least 660 times larger than the one of
nuclear spin transitions. In ENDOR experiments, part of this polarization is transferred to the
nuclear transitions, so that these transitions can be detected with much higher sensitivity. The
detection is also performed on electron spin transitions. Because of the higher energy of mw
photons compared to rf photons, this results in an additional sensitivity gain.
For liquid samples, ENDOR is best performed as a cw experiment. As cw ENDOR
involves simulataneous detection and strong mw and rf irradiation, it is a technically
demanding experiment. It is often easier to analyze the more complicated EPR spectra or to
perform a pulsed ENDOR experiment on a frozen solution, which yields additional
information on the anisotropy of the hyperfine couplings. For solid samples, pulsed ENDOR
experiments have replaced cw ENDOR almost completely, as they are technically less
demanding and work over a broader range of temperatures. Therefore, only pulsed ENDOR is
discussed in this lecture course.
Figure 10.22: Pulse sequence of the Davies ENDOR experiment. The influence of a rf t pulse with
variable frequency on echo inversion is observed. The echo is formed and inverted by selective mw
pulses with an excitation bandwidth that is smaller than the hyperfine coupling. Level populations at
points 0 (thermal equilibrium), 1 (after inversion), and 2 (after the rf pulse) are shown in Fig. 10.23.
The conceptually most simple ENDOR experiment is Davies ENDOR (Fig. 10.22). In
this experiment one of the two electron spin transitions of a hyperfine multiplet is selectively
inverted by an mw t pulse. The influence of this inversion pulse on level populations for the S
= 1/2, I = 1/2 system is shown in Fig. A,B. The pulse creates a state of two-spin order. An
p
p/2 p
p
t t T
m.w.
r.f.
0 1
2
216

ideally selective mw pulse creates polarization with opposite sign on the two individual
nuclear spin transitions. This polarization is just as large as the thermal equilibrium
polarization of the electron spin transitions.
Figure 10.23: Polarization transfers in the Davies ENDOR experiment for an S = 1/2, I = 1/2 system.
A) Thermal equilibrium populations corresponding to point 0 in the pulse sequence shown in Fig.
10.22. Only the electron transitions are significantly polarized. B) The selective mw pulse has
generated a state of two-spin order, where both nuclear transitions are oppositely polarized (point 1 in
the pulse sequence). C-E) Situations corresponding to point 2 in the pulse sequence for different
frequencies of the selective rf pulse. C) Situation after an rf pulse that is resonant with the
transition. Both electron transitions are saturated, i.e. devoid of polarization. D)
Situation after a non-resonant rf pulse. The originally excited electron transition is still inverted. E)
Situation after an rf pulse that is resonant with the transition. Both electron transitions
are saturated.
The rf t pulse is also transition-selective. It inverts either the transition
(Fig. 10.23C) or the transition (Fig. 10.23E) or it is off-resonant (Fig. 10.23D). In
the cases where the rf pulse is resonant with one of the nuclear transitions, both electron
transitions become saturated. In this situation the subsequent detection echo subsequence,
consisting of selective mw t/2 and t pulses on transition , does not give rise to an
echo (dotted zero line in Fig. 10.22). For an off-resonant rf pulse the transition is
still inverted and a negative echo is observed (solid signal line).
A Davies ENDOR spectrum of a single crystal of a copper(II) complex diluted into the
corresponding nickel(II) complex is shown in Fig. 10.24A. The lines are due to protons with
| aa
| ba
| ab
| bb
MW
RF
RF
A B
C
D
E
oo ' , o| ' ,
|o ' , || ' ,
oo ' , o| ' ,
|o ' , || ' ,
|o ' , oo ' ,
|o ' , oo ' ,
217
small hyperfine couplings (arrows) and directly coordinated nitrogens with large hyperfine
couplings. Note that unlike integral NMR line intensities, ENDOR line intensities are not
proportional to the number of observed nuclei. This is because the rf field amplitude is not
constant over such a broad frequency range. Furthermore the rf field at the nucleus is enhanced
by the hyperfine coupling. This hyperfine enhancement results from the non-resonant adiabatic
motion of the magnetic moment of the electron spin induced by the rf field.
Figure 10.24: Davies ENDOR spectra of [(2,2-bipyridylamine)(diethylenetriamine)Cu]
2+
diluted into
a single crystal of the corresponding isomorphous Ni(II) complex obtained at a temperature of 10 K. A)
Lengths of the mw pulses t
t
(1)
= 400 ns, t
t/2
= 200 ns, t
t
(2)
= 400 ns, both proton and nitrogen
transitions are observed, arrows indicate proton transitions; B) Lengths of the mw pulses t
t
(1)
= 20 ns,
t
t/2
= 100 ns, t
t
(2)
= 200 ns, most of the proton transitions are suppressed. C) Structure of the complex
(adapted from: C. Gemperle, A. Schweiger, Chem. Rev. 1991, 91, 1481-1505).
Polarization transfer in Davies ENDOR crucially depends on the selective excitation of
one electron spin transition. The excitation bandwidth of the mw pulses thus has to be smaller
than the hyperfine coupling. With decreasing hyperfine coupling excitation thus has to be more
and more selective, so that the experiment looses sensitivity. Davies ENDOR is thus best
suited to detect nuclei with relatively large hyperfine couplings (5-50 MHz).
The suppression of signals with small hyperfine couplings can be described by a
selectivity parameter
, [10.45]
A
C
B
15 10 5 20
n
rf
(MHz)
Cu
N
HN
NH
N
H
N
HN
n
S
A
eff
t
t
1 ( )
2t
------------------ =
218

where A
eff
is the hyperfine splitting and the length of the first mw t pulse. Maximum
absolute ENDOR intensity V
max
is obtained for . As a function of n
S
, the absolute
ENDOR intensity is given by
. [10.46]
Eq. [10.46] describes the hyperfine contrast selectivity that can be used to edit spectra. By
shortening the mw pulse length from 400 to 20 ns in the example shown in Fig. 10.24
ENDOR lines from the weakly coupled protons are strongly suppressed.
Figure 10.25: Effect of selective irradiation of an inhomogeneously broadened spectral line. A) The
inhomogeneously broadened line is a superposition of lines of spin packets with a smaller
homogeneous linewidth determined by T
2
and different resonance offsets. B) The spin packets can be
individually excited, so that a hole is burnt into the spectral line. The minimum width of the hole for
very weak irradiation is the homogeneous linewidth 2/T
2
. For pulsed irradiation, the width of the whole
is determined by the maximum of 2/T
2
and the excitation bandwidth of the pulse.
The suppression effect can be understood by considering the polarization changes in an
inhomogeneously broadened EPR line. Such a line with width I
inh
consists of many
superimposed lines of spin packets with different resonance frequency (Fig. 10.25). Each of
these lines has a homogeneous linewidth . With weak continuous mw
irradiation it is possible to saturate just one spin packet and burn a hole with width I
hom
into
the line. An mw t pulse affects spin packets within its excitation bandwidth. Sensitivity of
pulsed EPR experiments increases with the number of excited spin packets.
A resonant rf pulse shifts half of the hole (half of the electron spin polarization) to a
transition whose resonance frequency differs by the hyperfine splitting A
eff
. This shift occurs
towards higher or lower frequencies, depending on which of the transitions or
was inverted by the mw t pulse. Thus half of the whole remains at zero
t
t
1 ( )
n
S
2 2 =
V n
S
( ) V
max
2n
S
n
S
2
1 2 +
----------------------
\ .
|
| |
=
t
t
1 ( )
G
inh
G
hom
G > 2/T
2
W W
n
mw
0
A B
I
hom
2 T
2
=
|o ' , oo ' ,
|| ' , o| ' ,
219
resonance offset, while a quarter is shifted by A
eff
and another quarter by -A
eff
(Fig. 10.26).
This shift of the hole can only occur if A
eff
is larger than the width I of the hole.
Figure 10.26: An on-resonant rf t pulse in Davies ENDOR shifts intensity from a hole burnt into the
inhomogeneous EPR line to side holes. A) Situation before the rf pulse (point 1 in Fig. 10.22). B)
Situation after the rf pulse (point 2).
10.4.3.2 Mims ENDOR
The inversion of spin packets can be performed in a more controlled way by splitting
the mw t pulse in two t/2 pulses that are separated by an interpulse delay t. This can be seen
by computing the effect of such a subsequence (t/2)
x
-t-(t/2)
x
on an inhomogeneously
broadened line with product operator formalism. For brevity, only the electron spin S is
considered so that the rotating-frame Hamiltonian is . The experiment starts at
thermal equlibrium with density operator .
1
Application of an ideal mw t/2 pulse
along x,
, [10.47]
results in
. [10.48]
After subsequent evolution for time t under the density operator is given by
. [10.49]
1
The negative sign is a consequence of the negative electron charge that leads to preferential population of
| states of electron spins with their spin antiparallel to the external field.
A B
A
eff
A
eff
W
W
G p 2 /t
p
H

0 O
S
S

z =
o

eq
S

z =
o

eq
o

t 2 S

x
o

1
S

y =
H

0
o

2
O
S
t ( )S

y cos O
S
t ( ) sin S

x =
220

The following t/2 pulse along x leaves the second term on the right-hand side of Eq. [10.49]
unaffected, while the first term is converted to negative polarization
. [10.50]
. The second term on the right-hand side of Eq. [10.50] decays by transversal relaxation or can
be removed by a phase cycle. The first term corresponds to a polarization grid in the
inhomogeneously broadened EPR line whose resolution depends on interpulse delay t, but not
on the excitation bandwidth (Fig. 10.27). The excitation bandwidth determines the envelope of
the grating. It is thus possible to create a finely spaced grid to which many spin packets
contribute.
Figure 10.27: Polarization grating in an inhomogeneous line created by a (t/2)
x
-t-(t/2)
x
subsequence
(numerical simulation). Unlike in the product operator treatment, the limited excitation bandwidth of
the mw pulses is considered in this simulation.
The polarization grid can be detected by applying another mw t/2 pulse. This creates
an FID signal of the oscillatory grating. As the Fourier transform of a cosine function is a Dirac
delta function, the FID signal corresponding to the first term on the right-hand side of Eq.
[10.50] is confined at time t. It is a stimulated echo signal. The width of the echo is determined
by the excitation bandwidth of the t/2 pulses and is thus comparable to the pulse length.
In the Mims ENDOR experiment the effect of an rf pulse with variable frequency on
the intensity of the stimulated echo is observed (Fig. 10.28). An on-resonant rf t pulse shifts
two quarters of the polarization grating by A
eff
and -A
eff
, respectively, just as the rf t pulse in
Davies ENDOR shifts two quarters of the burnt hole (compare Fig. 10.26). The unshifted half
of the grating and the two shifted quarters interfer. For with
interference is destructive. The stimulated echo is canceled. For an on-resonant rf pulse and
, the shifted gratings interfer constructively and the stimulated echo is nearly
o

3
O
S
t ( )S

z cos O
S
t ( ) sin S

x =
DW
1/t
A
eff
t 2k 1 + ( )t = k 0 1 . . . =
A
eff
t 2kt =
221
unaffected. This situation cannot be distinguished from the one for an off-resonant rf pulse.
The Mims ENDOR experiment thus features blind spots at ..
Figure 10.28: Pulse sequence for the Mims ENDOR experiment. The intensity of the stimulated echo
is observed as a function of the frequency of the rf t pulse. The density operators are given in
Eq. [10.47-10.50] with .
The ENDOR efficiency
[10.51]
quantifies this blindspot behavior. The total intensity of the stimulated echo scales with
. By considering this and Eq. [10.51], it can be shown that very small hyperfine
couplings are detected with highest sensitivity at t = T
2
. To safely detect all large couplings,
Mims ENDOR spectra have to be recorded at different t and added. This averages the blind
spots.
10.4.4 ESEEM spectroscopy and the HYSCORE experiment
In NMR spectroscopy spin echo signals usually decay smoothly as a function of the
interpulse delays. In EPR spectroscopy, the decay is often superimposed by modulations with
nuclear frequencies. This electron spin echo envelope modulation (ESEEM) effect allows for a
detection of nuclear spectra without directly exciting the nuclear spins. The indirect excitation
of the nuclear spins arises from the tilt of the effective field at the nucleus with respect to the
external field (Fig. 10.18). Excitation of coherence on formally forbidden transitions can be
understood by transforming the mw Hamiltonian to the eigensystem of the static Hamiltonian
given in Eq. [10.40].
A
eff
t 2kt =
p/2 p/2 p/2
t t T
p
m.w.
r.f.
s
0
^
s
1
^
s
2
^
s
3
^
o

1
.o

3
o

0
o

eq
=
F
ENDOR
1
4
--- 1 A
eff
t ( ) cos ( ) =
e
2t T
2

222

10.4.4.1 Transition moments for allowed and forbidden transitions
In Eq. [10.40] the static Hamiltonian of the S=1/2, I=1/2 spin system is given in a
system where the z axes of both the electron and nuclear spin frame are parallel to the external
magnetic field and perpendicular to the linearly polarized mw field. In this frame the
oscillatory mw Hamiltonian, which describes the excitation, is given by
, [10.52]
with . The effective g value g
eff,x
pertains to the x direction of the mw
field.
The spin transitions are, however, defined between eigenstates of the spin system. To
compute the excitation strengths for these transitions, Eq. [10.52] thus has to be transformed to
the eigenframe of . The necessary transformations can be read off directly from Fig. 10.18.
To bring the effective field axis to the z axis, the o subspace of the electron spin S has to be
rotated clockwise about the y axis of the nuclear frame by angle n
o
. This is described by a
unitary transformation
, [10.53]
where n
o
is defined as
. [10.54]
The polarization operator is given by . The negative sign of the argument
of the arcustangens arises since a clockwise rotation is opposite to a rotation in mathematical
sense. A clockwise rotation is required since the y axis of a right-handed frame points into the
paper plane in Fig. 10.18. Likewise, the subspace corresponding to the | state of S has to be
rotated anticlockwise about the y axis of the nuclear frame by angle n
|
. This is described by a
unitary transformation
[10.55]
with
H

0
H

1 e
1
S

x =
e
1
g
eff x .
u
B
B
1
h =
H

0
U
o
in
o
S

o
I

y ( ) exp =
n
o
arc
B
2e
I
A +
-------------------
\ .
| |
tan =
S

o
S

o
E

2 S

z + =
U
|
in
|
S

|
I

y ( ) exp =
223
, [10.56]
and the polarization operator . In this frame the static Hamiltonian for the
weak coupling case
1
takes the form
. [10.57]
Since and commute, the transformation from Eq. [10.40] to Eq. [10.57] can be
described by a single unitary transformation
. [10.58]
The transformation of Eq. [10.52] to the eigensystem of is more simple with
Cartesian operators. By substituting the polarization operators, U
EB
takes the form
, [10.59]
with
, . [10.60]
Since , the second term in the argument on the right-hand side of Eq. [10.59] has
no effect on . In the eigenbasis, the oscillatory Hamiltonian takes the form
. [10.61]
The first term on the right hand side of Eq. [10.61] describes excitation of the allowed
transitions. The second term is more easily interpreted when the product operator is written in
terms of ladder operators,
. [10.62]
1
In the strong coupling case, the sign of either the e
o
or the e
|
term changes. Which sign changes depends
on the relative signs of e
I
and A.
n
|
arc
B
2e
I
A
-------------------
\ .
| |
tan =
S

|
E

2 S

z =
H

0 O
S
S

z e
o
S

o
I

z e
|
S

|
I

z + + =
S

o
I

y S

|
I

y
U
EB
U
o
U
|
U
|
U
o
i n
o
S

o
I

y n
|
S

|
I

y + ( ) ( ) exp = = =
H

0
U
EB
i n2S

zI

y cI

y + ( ) ( ) exp =
n
n
o
n
|

2
------------------- = c
n
o
n
|
+
2
------------------- =
I

y S

x [ , ] 0 =
H

1
H

1
EB ( )
U
EB
H

1U
EB

n ( )e
1
S

x
cos n ( ) sin e
1
S

yI

y + = =
S

yI

y
1
4
--- S

+
I

-
S

-
I

+
S

+
I

+
S

-
I

-
+ ( ) =
224

The terms on the right hand side of Eq. [10.62] drive the forbidden electron-nuclear transitions
(first two terms) and (last two terms) as indicated in Fig. 10.29A.
Figure 10.29: Level scheme and schematic EPR spectrum of the S=1/2, I=1/2 system. A) Level scheme
with allowed electron (green), forbidden electron-nuclear (red), and nuclear (blue) transitions. B)
Spectrum with allowe (green) and forbidden (red) transitions, where and
The transition moment of allowed transitions thus scales with cosn, while the transition
moment of forbidden transitions scales with sinn. Spectral intensities are proportional to the
transition probability, which is the square of the transition moment. This is because the
transition moment applies to both excitation and detection. The EPR spectrum of the S=1/2,
I=1/2 system with anisotropic hyperfine interaction thus has the appearance shown in Fig.
10.29B. The hyperfine splitting is given by the difference frequency
[10.63]
and the splitting of the forbidden transitions by the sum frequency
. [10.64]
10.4.4.2 Two-pulse ESEEM
Two-pulse ESEEM is observed by measuring the amplitude of a (t/2)-t-(t)-t echo as a
function of the interpulse delay t. The first t/2 pulse excites coherence with amplitude cosn on
the two allowed transitions and coherence with amplitude sinn on the two forbidden
transitions. The coherence evolves during the first interpulse delay t, defocuses due to the
distribution of resonance offsets O
S
, and decays with the transversal relaxation times T
2a
or T
2f
of the respective transitions. The t pulse inverts the phase of all coherences and thus leads to a
o| ' , |o ' , oo ' , || ' ,
1
2
3
4
w
a
w
b
w
24
w
13
w
14
w
23
w
w
W
s
+
aa
ab
ba
bb
A B
cos
2
h
sin
2
h
e
-
e
o
e
|
=
e
+
e
o
e
|
+ =
A
eff
e
-
e
o
e
|
= =
e
+
e
o
e
|
+ =
225
refocusing of magnetization after another delay t. Those pathways, where the coherence
remains on the same transition during the transfer by the t pulse, contribute two-pulse echoes
that smoothly decay with the respective relaxation rates.
Figure 10.30: Branching of magnetization (coherence transfers) during the t pulse in the two-pulse
ESEEM experiment. A) Transfer of coherence on the allowed transition to the two allowed
and two forbidden transitions combined with a phase inversion. B) Transfer of coherence on the
forbidden transition to the two allowed and two forbidden transitions combined with a phase
inversion.
However, the t pulse also changes the nuclear spin states with a probability sin
2
n, and
this corresponds to a transfer of coherence between the different transitions. This branching of
magnetization is indicated in a density matrix representation in Fig. 10.30. For instance,
coherence on the allowed transition is merely phase-inverted with probability
cos
2
n and transferred with probability sin(n)cos(n) to the forbidden transition (Fig.
10.30A). In contrast, coherence on the forbbiden transition is phase-inverted only
with the small probability sin
2
n and transferred to the forbidden transition with the
larger probability cos
2
n (Fig. 10.30B).
All transferred coherences also experience phase inversion. Thus they also form
echoes. These coherence transfer echoes oscillate with the difference of the transition
frequencies before and after the t pulse. From Fig. 10.29 and Eqs. [10.63] and [10.64] it can be
verified that the possible difference frequencies are e
o
, e
|
, e

, and e
+
. Each of the basic
nuclear frequencies e
o
and e
|
appears in two coherence transfer pathways, and each of the
combination frequencies e

and e
+
appears in only one pathway. Furthermore, the product of
the excitation probability (transition moment for excited coherence), transfer probability
(branching factor), and detection probability (transition moment for detected coherence) is the
A B
cos h
cos h
cos h
cos h
cos h
-sin h
sin h
cos h
-sin h
sin h sin h
sin h
2
2
2
2
|aa
aa|
ab|
ba|
bb|
|aa |ab |ab |ba |ba |bb |bb
oo |o
oo ||
oo |o
oo ||
oo ||
o| |o
226

same for each pathway, it takes the value sin
2
n cos
2
n = sin
2
(2n)/4. The amplitude of all
nuclear modulations thus scales with the modulation depth parameter
. [10.65]
With proper bookeeping of the coherence transfer pathways, the two-pulse ESEEM formula
[10.66]
results. A schematic spectrum for the weak coupling case is shown in Fig. 10.31. Note that he
combination frequencies are close to A and 2e
I
, but are slightly shifted towards lower and
higher frequencies, respectively. The phase information (positive basic frequency peaks and
negative combination peaks) is often hard to access, as spectrometer dead time prevents
observation of the signal at short t values. Usually phase correction fails and magnitude
spectra are displayed, where all peaks are positive.
Figure 10.31: Schematic two-pulse ESEEM spectrum for a weakly coupled S=1/2, I=1/2 system.
10.4.4.3 Three-pulse ESEEM
Nuclear frequencies are measured by two-pulse ESEEM as differences between the
frequencies of electron spin transitions. Hence, the linewidth in the spectra is twice the
homogeneous EPR linewidth, which is much larger than the natural linewidth for nuclear
k 2n sin
2
Be
I
e
o
e
|
--------------
\ .
| |
2
= =
V
2p
t ( ) 1
k
4
--- 2 2 e
o
t ( ) cos 2 e
|
t ( ) cos e
-
t ( ) cos e
+
t ( ) cos + + ( ) =
A
w
I
2w
I
227
transitions. The resolution can thus be drastically improved by observing the evolution of
coherence on nuclear transitions.
Figure 10.32: Magnetization branching during the second t/2 pulse in a mw pulse subsequence
. A) Coherence on the allowed electron spin transition is transferred
to nuclear coherences with probability sinn. B) Coherence on the forbidden transition is
transferred to nuclear coherences with probability cosn.
A single, ideal mw pulse does not excite nuclear coherence. However, an mw pulse
subsequence (t/2)-t-(t/2) does generate such coherence due to magnetization branching
during the second t/2 pulse (Fig. 10.32). This nuclear coherence evolves during delay T and is
then transferred to observable electron coherence by another t/2 pulse. After another delay t,
the dispersion of electron spin resonance offsets O
S
is refocused. The coherence transfer
pathways that involve nuclear coherence contribute signals that oscillate with frequencies e
o
and e
|
as a function of time T. Coherence transfer pathways that do not involve nuclear
coherence give rise to a smoothly decaying stimulated echo. The total echo signal as a function
of the fixed interpulse delay t and the variable delay T, neglecting relaxation, is given by
. [10.67]
No oscillation with combination frequencies is observed. This simplifies spectra and is an
additional advantage of three-pulse ESEEM compared to two-pulse ESEEM. However, the
amplitude of the nuclear oscillation now depends on the nuclear frequency in the other electron
spin state and on the fixed delay t. This leads to blind spots, which are a disadvantage of three-
A B
|aa
aa|
ab|
ba|
bb|
|aa |ab |ab |ba |ba |bb |bb
cosh
cosh
cosh
sinh
cosh
i
2
i
2
sinh
sinh
i
2
i
2
i
2
i
2
-
i
2
-
sinh
i
2
-
t 2 ( ) t t 2 ( ) oo |o
oo ||
V
3p
t T . ( ) 1
k
4
--- 1 e
|
t ( ) cos | | 1 e
o
T t + ( ) ( ) cos | | { =
1 e
o
t ( ) cos | | 1 e
|
T t + ( ) ( ) cos | | } +
228

pulse ESEEM. To avoid suppression of peaks, the experiment thus has to be performed at
several t values and the magnitude spectra have to be added.
Figure 10.33: Pulse sequence for three-pulse ESEEM. The echo amplitude is observed as a function of
interpulse delay T for fixed delays t.
10.4.4.4 HYSCORE
Hyperfine sublevel correlation spectra, as introduced in Section 10.4.2.3, can be
measured by a two-dimensional extension of the three-pulse ESEEM experiment. For that
purpose, an mw t pulse is introduced at a variable delay t
1
after the second t/2 pulse (Fig.
10.34). This t pulse transfers nuclear coherence between the two manifolds corresponding to
the electron spin states o and |. Thus it correlates frequency e
o
of a given nucleus with
frequency e
|
of the same nucleus and vice versa. The new frequency is measured by
introducing another variable delay t
2
between the t pulse and the final t/2 pulse that
reconverts the nuclear coherence to observable electron coherence.
Figure 10.34: Pulse sequence of the two-dimensional HYSCORE experiment. Echo amplitude is
observed as a function of the two variable delays t
1
and t
2
for fixed delays t.
Forbidden transitions during the t pulse convert some nuclear coherence to electron
polarization and vice versa. This leads to axial peaks at e
1
= 0 and e
2
= 0. These peaks are
unwanted, as they do not contain correlation information. They can be removed by baseline
correction of the time-domain data before Fourier transformation. Because of its limited
excitation bandwidth, the nominal t pulse does not fully invert the electron spin for large
resonance offsets O
S
. This leads to diagonal peaks e
1
= e
2
= e
o
and e
1
= e
2
= e
|
. These
p/2 p/2 p/2
t t T
p/2 p/2 p/2
p
t t t
1
t
2
229
diagonal peaks cannot reliably be removed by data processing and may obscure cross peaks for
small hyperfine couplings. Their contribution scales with the ratio between the excitation
bandwidths of the t/2 pulses and the t pulse. Therefore, it is advantageous to use the full mw
power for the t pulse and less power for the t/2 pulses, as indicated in Fig. 10.34.
The modulation in the HYSCORE experiment corresponding to the cross peaks is
described by
[10.68]
with
,[10.69]
.[10.70]
In this representation with unsigned nuclear frequencies, the weak coupling case corresponds
to and the strong coupling case to . In the former case, the cross
peaks are thus stronger in the quadrant (e
1
>0, e
2
>0) and in the latter case in the quadrant
(e
1
>0, e
2
<0).
The blind spot behavior of the HYSCORE experiment is described by the two factors
and in Eq. [10.68]. A cross peak is thus suppressed when one of the
two correlated nuclear peaks is suppressed in three-pulse ESEEM. Therefore it is even more
important in HYSCORE to add several magnitude spectra obtained at different delays t.
10.5 Spin probes, spin labels, and spin traps
Structure and dynamics of complex biological systems or synthetic materials are often
difficult to characterize by techniques that derive their signal from the whole sample. The
problems are a lack of contrast between different components, insufficient resolution, and
ambiguities in signal assignment. The situation simplifies considerably with techniques that
detect signals only from a probe molecule that is attached to a certain site of interest in the
system. EPR spectroscopy can be used as such a probe technique for diamagnetic systems. The
V
4p
t t
1
t
2
. . ( )
k
2
---
e
o
t
2
----------
\ .
| |
sin
e
|
t
2
---------
\ .
| |
V
o| ( )
t t
1
t
2
. . ( ) V
|o ( )
t t
1
t
2
. . ( ) + | | sin =
V
o| ( )
t t
1
t
2
. . ( ) n cos
2
e
o
t
1
e
|
t
2
e
+
t
2
--- + +
\ .
| |
cos n sin
2
e
o
t
1
e
|
t
2
e
-
t
2
--- +
\ .
| |
cos =
V
|o ( )
t t
1
t
2
. . ( ) n cos
2
e
|
t
1
e
o
t
2
e
+
t
2
--- + +
\ .
| |
cos n sin
2
e
|
t
1
e
o
t
2
e
-
t
2
--- +
\ .
| |
cos =
n cos
2
n sin
2
> n sin
2
n cos
2
>
e
o
t 2 ( ) sin e
|
t 2 ( ) sin
230

spectroscopic response of the spin probe is interpreted in terms of structure and dynamics of
the system. Ideal probes do not perturb the system under investigation, can be directed at will
to sites of interest, and give a spectroscopic response that strongly depends on their
environment.
The EPR spin probe technique is particularly suitable for soft organic matter, as it does
not rely on long-range order and as the spectra of typical spin probes are very sensitive to
molecular dynamics on time scales between 10 ps and 1 us. This time range corresponds to
motion on length scales longer than a chemical bond, but smaller than colloidal dimensions
that start at about 100 nm. On these length scales supramolecular interactions determine the
self-organization, stability, and functionality of soft matter systems. Distances between spin
probes can be measured on length scales between about 0.8 and 8 nm.
Figure 10.35: Structures of the most common spin probes and labels. R is a functional group used for
dierctiong the probe or attaching the label to the site of interest. 1 4-oxo-2,2,6,6-tetramethyl-piperidin.
2 2,2,6,6-tetramethyl-piperidin-1-oxyls, TEMPO derivatives. 3 2,2,5,5-tetramethyl-pyrrolydine-1-
oxyls, PROXYL derivatives. 4 2,2,5,5-tetramethyl-pyrroline-1-oxyls. 5 4,4,-dimethyl-3-
oxazolidinyloxy probes, DOXYL derivatives. 6 Methanethiosulfonate spin label, MTSSL.
The most widely applied class of spin probes are nitroxides (Fig. 10.35). They are
originally derived from compound 1, 4-oxo-2,2,6,6-tetramethyl-piperidin, which is the basis
compound of hindered amine light stabilizers. This compound is formed in a one-pot reaction from
acetone and ammonia and is easily oxidized to TEMPON, 4-oxo-2,2,6,6-tetramethyl-piperidin-1-oxyl
(compound 2 with -R being =O). The carbonyl group can be converted to other functional groups (e.g. -
OH, -NH
2
, -N
+
(CH
3
)
3
). Such groups direct site attachment via hydrogen bonds or electrostatic
interactions (spin probes in a strict sense). Reactive groups R can be used for covalent attachment to the
site of interest (spin labels). Somewhat smaller probes or labels are obtained by reducing the ring size to
N
O
R
N
H
O
N
O
R
N
O
R
O N O
N O
S
S
O
O
H
3
C
1
5 6
2 3 4
231
PROXYL derivatives 3 or 3,4-dehydro-PROXYL derivatives 4. The most important label is the
methanethiosulfonate spin label (MTSSL) 6 that attaches with high selectivity under very mild
conditions to cysteine residues in proteins. The size of the MTSSL-derived sidechain is similar to the
one of an aromatic amino acid residue. The SDSL technique for proteins involves mutation of an amino
acid at the site of interest to cystein and subsequent reaction with a thiol-selective spin labels such as
MTSSL, iodoacetamido-PROXYL, or maleimido-PROXYL. For distance measurements two selected
sites are labeled.
Synthesis of DOXYL derivatives 5 is more difficult and proceeds with lower yields.
Their advantage is a very rigid attachment to alkyl chains that allows to probe motion and
liquid crystalline ordering of surfactant molecules, lipids, and steroids in more detail.
The unpaired electron in nitroxides is stabilized by delocalization over the N-O bond
and by the steric hindrance due to the four methyl groups that prevents dimerization.
Derivatives of TEMPO, PROXYL, and 3,4-dehydro-PROXYL are usually stable up to
temperatures of 130 C. They are degraded by strong acids (pH 1 and below) and slowly
decompose in solution at pH 2 and below. Nitroxides are reduced to the corresponding
hydroxylamines by ascorbic acid and may be reduced by some thiols. PROXYL derivatives
are less susceptible to this reduction than TEMPO derivatives. The hydroxylamines are
reoxidized by air.
Figure 10.36: Molecular frame (PAS of the g tensor) for nitroxides and dependence of the
14
N
hyperfine coupling on orientation of the magnetic field with respect to this frame.

H
N
R
O
z
y
x
A( N)
14
A( N)
14
232

10.5.1 Nitroxide spectra with and without orientational averaging
The unpaired electron in nitroxides is distributed mainly in a t orbital along the N-O
bond that is made up of the p
z
orbitals of the nitrogen and oxygen atom. There is also some
spin density in the s orbitals of these two atoms, but few delocalization to the neighboring
carbon atoms. The deviation of the g value from g
e
is due to spin orbit coupling in excited
states that involve the lone pair orbitals on oxygen. This deviation is maximum in the x
direction of the molecular frame along the N-O bond (Fig. 10.36), intermediate in the y
direction, and almost negligible in the z direction. Typical principal g values are g
xx
= 2.0090,
g
yy
=2.0060, and g
zz
= 2.0024. In contrast, the hyperfine coupling to
14
N is strongest if the
external field is along the z direction, i.e. parallel to the lobes of the p
z
orbital on nitrogen.
Differences between the hyperfine coupling in the x and y direction are minute, so that an
axially symmetric hyperfine tensor can be assumed. Typical hyperfine couplings are A
xx
= A
yy
= 18 MHz, A
zz
= 96 MHz. All magnetic parameters differ slightly between the different classes
of nitroxides. The z axes of the hyperfine and g tensor are nearly coincident. Hyperfine
couplings to the protons of the methyl groups in the 2- and 6-positions may be resolved in the
limit of fast motion (see Section 10.5.2) in the absence of oxygen. In other cases the proton
couplings are unresolved and lead to line broadening. Narrower lines are then obtained with
deuterated nitroxides.
Figure 10.37: Schematic energy level scheme and cw EPR spectrum of a nitroxide radical. Only the
electron Zeeman and
14
N hyperfine interactions are considered.
The EPR spectrum of a nitroxide in a single orientation or in the regime of fast
orientational averaging is schematically shown in Fig. 10.37. The energy levels of the unpaired
electron result from electron Zeeman splitting in the external field (magnetic quantum number
E
B
0
+1/2
-1/2
hn
mw
-1
0
+1
+1 0 -1
+1
0
-1
m
S m
I
233
m
S
) and hyperfine coupling to the
14
N nuclear spin (product of quantum numbers m
S
and m
I
).
For the allowed transitions m
I
is constant, so that a hyperfine triplet results. As the hyperfine
coupling is positive, the transition with the highest frequency corresponds to m
I
= +1. In a field
sweep this transition is detected at lowest field. When Fig. 10.37 is interpreted as a spectrum in
the regime of fast orientational averaging, the center line is at a field corresponding to the
isotropic g value g
iso
= (g
xx
+g
yy
+g
zz
)/3 and the splitting between two outer lines is 2A
iso
=
2(A
xx
+A
yy
+A
zz
)/3. If the Figure is interpreted as a single-crystal spectrum, the center line is
positioned at the g value along the magnetic field direction in the molecular frame and the
splitting is determined by the hyperfine coupling along this direction.
The powder spectrum is the superposition of the spectra at all orientations with proper
weighting. Each of the lines with different
14
N magnetic quantum number m
I
can be
considered separately (Fig. 10.38A). The variation of the resonance field is minimum for the
m
I
= 0 transition, where the hyperfine contribution vanishes. It is maximum for the m
I
= -1
transition, where the hyperfine contribution and electron Zeeman contribution have the same
sign, and intermediate for the m
I
= +1 transition, where the two contributions have different
sign.
Figure 10.38: Nitroxide spectrum for a macroscopically disordered system at X band frequencies. A)
Absorption subspectra of the three transitions with different
14
N nuclear magnetic quantum number m
I
.
B) Total absorption spectrum, resulting from the sum of the three subspectra (top) and its derivative
(bottom) that corresponds to the cw EPR spectrum.
The total absorption spectrum is the sum of the two subspectra (Fig. 10.38B). It can be
measured by echo-detected field-swept EPR. In cw EPR the derivative of this absorption
spectrum is observed. It has a positive peak at the low-field edge that corresponds to the edge
of the m
I
= +1 line and a negative peak at the high-field edge that corresponds to the edge of the
m
I
= -1 line. The outer extrema splitting between these two peaks is twice the hyperfine
0.34 0.34 0.345 0.345 0.35 0.35
m
I
=-1
m
I
=0
m
I
=+1
B
0
(T) B
0
(T)
A B
2A
zz
234

coupling along the molecular z orientation 2A
zz
. The central line with a quasi-dispersive shape
results mainly from the m
I
= 0 transition.
10.5.2 Nitroxide spectra for incomplete orientational averaging
The effect of rotational motion of the nitroxide on the spectrum can be understood in
terms of a multi-site exchange between different orientations. The exchange rate is related to
the rotational diffusion rate R, which in turn is given by R= 1/6t
r
, where t
r
is the rotational
correlation time. The basic concepts are the same as for two-site chemical exchange (Sections
3.1 and 3.2). In the limit of slow exchange, the spectrum is lifetime broadened (Figure 3.8).
This effect is unresolved in nitroxide spectra. If the motion becomes faster, exchange between
orientations in addition to the line broadening leads to partial averaging of the two resonance
frequencies (Fig. 3.6). As a consequence the outer extrema splitting 2A
zz
reduces (Fig. 10.39).
This effect becomes observable at rotational correlation times shorter than about 1 us.
Figure 10.39: Dependence of the nitroxide cw EPR spectrum on the rotational correlation time t
r
.
Fast regime Slow tumbling
2 3.0 mT A '
zz

2 6.8 mT A '
zz

t
r
t
r
10 ps 4 ns
32 ps
10 ns
100 ps
32 ns
316 ps 200 ps
1 ns 316 ns
3 ns
1 s
235
For a symmetric two-site exchange a normalized exchange rate constant of
corresponds to coalescence, i.e. a collapse of the doublet into a single line, and maximum
exchange broadening (Fig. 3.6). For the multi-site orientational exchange in nitroxides such
coalescence is observed at a rotational correlation time of about 3.5 ns, where the appearance
of the spectrum changes from a powder like lineshape (right column in Fig. 10.39, slow
tumbling) to a triplet of Lorentzian broadened lines (left column, fast regime). In the fast
regime the outer extrema splitting 2A
zz
is only slightly larger than twice the isotropic
hyperfine coupling (Fig. 10.40). The outer extrema splitting depends most strongly on t
r
at
coalescence, where it is mT. Dynamic processes are often studied as a function of
temperature. They can then be characterized by the temperature T
5mT
(or T
50G
), where
mT is attained.
Figure 10.40: Dependence of the outer extrema splitting 2A
zz
on the rotational correlation time (semi-
logarithmic plot).
For thermally activated processes, 1/t
r
is expected to have an Arrhenius dependence on
temperature,
, [10.71]
where E
A
is the activation energy. The rotational correlation times at different temperatures
can be determined by lineshape fitting.
These considerations assume isotropic Brownian rotational diffusion, which is often a
good approximation for small, almost spherical spin probes like TEMPO (compound 2, R=H).
Different lineshapes are observed for more complicated dynamic processes. However, only in
simple cases it is possible to derive a model for the motion from the spectral lineshape.
1 2 2 ( )
2A'
zz
5 ~
2A'
zz
5 =
-10 -9 -8 -7 -6
3
4
5
6
7
2
'
(
m
T
)
A
z
z
log( /s) t
r
slow
tumbling
fast
regime
1 t
r
( ) ln A E
A
RT ( ) + =
236

Lineshapes predicted from molecular dynamics simulations are often in good agreement with
experimental lineshapes.
In the fast regime, the widths of the three lines are different. This can be understood
from Fig. 3.9. The mean square difference of the resonance frequencies of
exchanging orientations is smallest for the narrow central line with m
I
= 0 and largest for the
broad high-field line with m
I
= -1. Therefore, the high-field line is broader also in the case of
fast orientational exchange. Only in the fast limit, where other contributions to line broadening
dominate, equal widths and amplitudes are observed for the three lines. This limit is attained at
rotational correlation times of 10 ps or shorter.
In the fast regime t
r
can be obtained from analyzing the linewidths AB according to
Kivelson theory.
1
According to this theory, the ratio of the line width of one of the outer lines
to the line width of the central line is given by
, [10.72]
where
[10.73]
and
, [10.74]
with the hyperfine anisotropy parameter
[10.75]
and the electron Zeeman anisotropy parameter A
. [10.76]
1
D. Kivelson, J. Chem. Phys. 1960, 33, 1094-1106. Theory of ESR Linewidths of Free Radicals.
O
1
O
2
( )
2
T
2
1
m
I
( )
T
2
1
0 ( )
-------------------- 1 Bm
I
Cm
I
2
+ + =
B
4
15
------ bAB
0
T
2
0 ( )t
r
=
C
1
8
-- -b
2
T
2
0 ( )t
r
=
b
4t
3
------ A
zz
A
xx
A
yy
+
2
------------------------- =
A
2tu
B
h
------------- g
zz
g
xx
g
yy
+
2
---------------------- =
237
The relaxation time T
2
(0) for the central line can be computed from the corresponding peak-to-
peak linewidth in field domain as
. [10.77]
Thus, Eqs. [10.72-10.74] can be solved for the only remaining unknown t
r
. In practice, ratios
of peak-to-peak line amplitudes I(m
I
) are analyzed rather than linewidth ratios, as they can be
measured with higher precision. The linewidth ratio is related to the amplitude ratio by
, [10.78]
since the integral intensity of the absorption line is the same for each of the three transitions.
The rotational correlation time can thus be determined by, e.g.,
, [10.79]
where AB
0
is the peak-to-peak linewidth of the central line. Similar formulas can be obtained
by using different pairs of line amplitudes or all three line amplitudes. It is good practice to test
whether several of these formulas give similar values for t
r
. If this is not the case, the motion
cannot be characterized by a single rotational correlation time.
10.5.3 Dependence of magnetic parameters on polarity and hydrogen bonds
The g and hyperfine tensor of a nitroxide depend on the distribution of the unpaired
electron between the orbitals on the nitrogen and oxygen atom. This distribution in turn is
influenced by the polarity of the environment, as can be understood from the mesomeric
structures shown in Fig. 10.41. Localization of the unpaired electron at the nitrogen atom
corresponds to a charge-separated structure that is stabilized by a polar environment. Therefore
more spin density p
N
on the nitrogen and less spin density p
O
on the oxygen atom is found in
such a polar environment.
AB
pp
0 ( )
T
2
0 ( )
h
t 3g
iso
u
B
AB
pp
0 ( )
------------------------------------------------ =
T
2
1
m
I
( )
T
2
1
0 ( )
--------------------
I m
I
( )
I 0 ( )
------------- =
t
r
t 3
b
----------
b
8
---
4AB
0
15
----------------
1
g
iso
u
B
h
----------------AB
pp
0 ( )
I 0 ( )
I 1 ( )
------------- 1 =
N N O O
+ -
238

Figure 10.41: Mesomeric structures of a nitroxide with the unpaired electron localized on the nitrogen
atom (left) and the oxygen atom (right).
The isotropic
14
N hyperfine coupling A
iso
and the coupling along the lobes of the p
z
orbital (A
zz
) increase with increasing p
N
and thus with increasing polarity. The deviation of the
g value from g
e
changes in the opposite way, as it results mainly from spin-orbit coupling in an
excited state that involves alone pair localized on oxygen. The strongest change is observed for
g
xx
, as is expected when the lone pair orbitals have mainly p
y
character.
1
The g value shift
Ag
xx
= g
xx
-g
e
is influenced not only by changes in the spin density distribution, but also by
changes in the energy difference between the SOMO and the lone pair orbital. Hydrogen
bonding lowers the energy of the lone pair and thus increases this energy difference. This
corresponds to a blue shift of the n-t* transition in optical spectra and to an increase of the
denominator in Eq. [10.8]. Thus hydrogen bonding leads to a smaller g shift Ag
xx
at same
polarity. Hydrogen bonding also leads to some delocalization of the lone pair orbitals, which
leads to a minor further decrease of Ag
xx
. By measuring both A
zz
and g
xx
with high precision
with high-field EPR, it is possible to separate the contributions due to polarity and hydrogen
bonding.
10.5.4 Accessibility measurements
The influence of Heisenberg exchange of unpaired electrons between paramagnetic
species on relaxation times (see Section 10.4.1, Fig. 10.12) can be used to estimate the local
concentration of a paramagnetic quencher near a nitroxide spin label. An ubiquitous
paramagnetic quencher is triplet oxygen, which is much better soluble in apolar environments,
such as lipid bilayers, than in polar environments. Water-soluble quenchers are transition metal
complexes such as the electroneutral Ni(II) complex of ethylendiaminediacetic acid (EDDA)
or the negatively charged Cr(III) complex of oxalate [Cr(C
2
O
4
)
3
]
3-
. At very high local
concentrations such quenchers cause line broadening. At lower concentration their influence
on relaxation times can be quantified easily and precisely by measuring saturation curves (see
Section 2.7.2).
1
Fo a full discussion, see: T. Kawamura, S. Matsunami, T. Yonezawa, Bull. Chem. Soc. Japan 1967, 40,
1111-1115. Solvent Effects on the g-Value of Di-t-butyl Nitric Oxide.
239
The change in relaxtion rates is purely through collisions (Fig. 10.11) if the
longitudinal relaxation time of the quencher is much shorter than the lifetime of the collisional
encounter complex. In this regime it is equal to the exchange rate W
ex
,
, [10.80]
where k
ex
ist the exchange rate constant and C the local quencher concentration.
1
In the
following we assume the case of insignificant exchange broadening, . Up to a
constant factor o, W
ex
can then be determined from saturation curves. For this, the peak-to-
peak amplitude A of the first derivative central line of the nitroxide spectrum (m
I
= 0) is
measured as a function of mw power P. The data are described by the function
[10.81]
with the power-independent amplitude factor I, the power at half saturation P
1/2
, and the
inhomogeneity parameter c as adjustable parameters. The inhomogeneity parameter takes the
value c= 1.5 in the homogeneous limit, which is assumed from here on, and c= 0.5 in the
inhomogeneous limit. More precisely, P
1/2
is the incident mw power where A is reduced to
half of its unsaturated value. This is given by
, [10.82]
where is the conversion efficiency of the mw resonator. For small quencher
concentrations exchange broadening is insignificant and T
2
is the same in the presence and
absence of the quencher. The change in P
1/2
due to addition of the quencher is then given by
. [10.83]
1
The term local refers to the characteristic diffusion length of the quencher on the time scale given by W
ex
.
The factorization into k
ex
and C may be ill-defined when comparing labels attached at different sites of a
protein, but W
ex
remains a meaningful parameter.
A
1
T
1
------
\ .
| |
A
1
T
2
------
\ .
| |
W
ex
k
ex
C = = =
W
ex
1 T
2

A P ( )
I P
1 2
1 c
1 ( )P P
1 2
+ | |
c
----------------------------------------------------------- =
P
1 2
4
3
1

e
2
A
2
T
1
T
2
------------------------- =
A B
1
P =
AP
1 2
4
3
1 ( )W
ex

e
2
A
2
T
2
-------------------------------- =
240

To eliminate the dependencies on T
2
and A, a dimensionless accessibility parameter H is
defined as
1
, [10.84]
where the denominator is obtained from measurements on a reference substance such as dilute
diphenylpicrylhydrazyl powder in KCl.
10.5.5 Spin trapping
In many processes that involve free radicals, these radicals react very fast, so that their
lifetimes are extremely short and their steady-state concentrations extremely low. Such
radicals can be detected indirectly by trapping them with a diamagnetic compound. The
reaction product is a stable free radical whose magnetic parameters provide some information
on the original, short-living radical. The most important class of diamagnetic spin traps are
nitrones, such as o-phenyl-t-butyl nitrone (PBN) or 5,5-dimethyl-1-pyrroline N-oxide
(DMPO). Free radicals add to the o carbon of nitrones and a nitroxide is formed (Fig. 10.42).
The hydrogen bound to the o carbon makes nitroxides generated by such reactions less stable
than the nitroxides used as spin probes. The lifetime of spin trap products in solution typically
ranges between a few seconds and a few hours. This is usually sufficient to accumulate steady-
state concentrations that are detectable by cw EPR
Figure 10.42: Reaction of the spin traps o-phenyl-t-butyl nitrone (PBN) or 5,5-dimethyl-1-pyrroline N-oxide
(DMPO) with free radicals R
1
C. Altenbach, W. Froncisz, R. Hemker, H. Mchaourab, W. L. Hubbell, Biophys. J. 2005, 89, 2103-2112.
Accessibility of Nitroxide Side Chains: Absolute Heisenberg Exchange Rates from Power Saturation EPR.
H
AP
1 2
AB
pp
0 ( )
AP
1 2
AB
pp
0 ( ) | |
ref
-------------------------------------------------- oW
ex
= =
N
N
O
O
N
N
O
O
PBN
DMPO
a
a
+R

R
R
+R
H
H
241
.
Figure 10.43: cw EPR spectrum observed by UV irradiation ( < 380 nm) of a dispersion of ZnO in
heptane in the presence of oxygen and the spin trap DMPO (from C. Cheng, R. P. Veregin, J. R.
Harbour, M. I. Hair, S. L. Issler, J. Tromp, J. Phys. Chem. 1989, 93, 2607-2609. Photochemistry of ZnO
in Heptane: Detection by Oxygen Uptake and Spin Trapping.
The hyperfine coupling to the |-proton is usually resolved, so that a six-line spectrum
results. In some cases, further couplings may be resolved. For instance, the spectrum of the
DMPO adduct of the superoxide anion radical O
2
-
(Fig. ) has twelve lines with couplings A
N
= 12.9 G, A
|
H
= 6.3 G, A

H
= 1.6 G. The coupling to one of the two -protons is not fully
resolved. Spin trapping has its main field of application in studies on degeneration processes in
living cells. Such processes involve reactive oxygen species, most of which are radicals.
Furthermore spin-trapping is used in studies on hetrerogeneous catalysts that promote radical
formation. The radical adducts of spin traps are mainly identified via the
14
N and o-
1
H
hyperfine couplings. The isotropic g value of the adduct also depends on the type of original
radical, but is less characteristic. An internet database for identification of radical adducts of
several widely used spin traps is maintained by the NIH (http://tools.niehs.nih.gov/stdb/
index.cfm).
242

10.6 Distance measurements by EPR techniques
The high sensitivity of EPR spectroscopy and the larger magnetic moment of the
electron spin allow for the measurement of longer distances between spins than is possible
with NMR. In conjunction with SDSL this allows for studying structure and structural
dynamics of biomacromolecules and their complexes, even if they cannot be crystallized and
have molecular weights larger than 100 kDa. The main current application field are large
membrane proteins. Similar techniques can also be applied to synthetic polymers or to soft
matter that forms self-organized structures based on supramolecular interactions.
Distance measurements are based on the inverse cube dependence of the magnetic
dipole interaction on distance (Section 5.8). The data can be analyzed with high precison
( for distances up to 40 ) if the spin pairs are sufficiently diluted and exchange
coupling (10.3.6.1) is negligible. On length scales between 0.8 and 8 nm, where such
measurements are possible, spins are sufficiently diluted at concentrations of 200 uM or less.
Exchange couplings are usually negligble at distances beyond 1.5 nm. Shorter distances than
1.5 nm can be determined with a precision of about .
10.6.1 cw EPR
10.6.1.1 Analysis of dipolar line broadening
The peaks in a well resolved first derivative absorption spectrum of a nitroxide in the
rigid limit have a typical width of 0.4 mT corresponding to 11.2 MHz. An additional
broadening with about half this width can be safely detected. According to Eq. [10.32] this
corresponds to a spin-spin distance of up to 21 that still leads to recognizable dipolar
broadening. If the spin label environment is homogeneous and very similar for both labels, the
peak width in cw EPR spectra is determined by unresolved hyperfine couplings of methyl
protons in the label. The limit can then be shifted to distances of about 25 by using a
deuterated label. If the environment is heterogeneous or different for the two labels,
differences in hydrogen bonding and polarity may cause additional broadening due to a
distribution of the
14
N hyperfine coupling and g value (Section 10.5.3). The upper limit for
0.5
2
243
elucidating accurate distances then decreases to about 16 and cannot be extended by
deuteration.
The distance can be determined with the highest precision if the cw EPR spectra both in
the absence and presence of the dipole-dipole interaction are available. Hence, the spectrum of
the doubly-labeled macromolecule and the two spectra of the corresponding singly-labeled
macromolecules have to be measured. The spectrum of the doubly labeled molecule can then
be fitted by convolution of the sum of the spectra of the isolated paramagnetic centers with a
dipolar broadening function (Fig. 10.44). The dipolar broadening function in turn is simulated
by superimposing Pake patterns for a Gaussian distribution of distances. Fit parameters are the
mean distance and width of the Gaussian peak.
Figure 10.44: Distance determination from a dipolar broadened cw EPR spectrum by fitting of a
convolution of the spectrum of isolated paramagnetic centers with a dipolar broadening function.
The convolution method assumes that the macromolecule is completely doubly
labelled. This assumption can be relaxed by fitting a superposition of the dipolar broadened
and unbroadened lineshapes. If the spectrum of the isolated centers is unknown, a simulated
spectrum can be used instead. Both these variations of the method cause a loss in accuracy and
a decrease in the upper distance limit.
10.6.1.2 Intensity of half-field transitions
The convolution technique neglects the influence of exchange coupling between the
electron spins on the line shape. This is a good approximation at the upper end of the distance
range (1.5... 2 nm), but a poor approximation at the lower end (0.8... 1.2 nm). The influence of
the isotropic exchange coupling is eliminated in a technique that relies on mixing of the states
r ( , o
r
isolated
centers
trial
distance
distribution
exp. dipolar
broadened
exp. dipolar
broadened
sim. dipolar
broadened
Integrate
FT
inverse FT
FT
Simulate/ fit
dipolar
spectrum
Multiply
derivative
Convolution
B
0
B
0
B
0
r
B
0
r
s
r
244

of the two spins by the anisotropic dipole-dipole coupling. The two types of electron-electron
coupling can be distinguished, since the anisotropic coupling contributes off-diagonal terms
that connect the and states of the S
1
= 1/2, S
2
= 1/2 two-spin system ( and terms
in the dipolar alphabet, see Eq. [5.80] and Fig. ), while the exchange coupling does not.
Therefore, only the dipole-dipole coupling mixes theses levels and leads to a non-zero
transition probability of the double quantum transition. At constant field, this
transition would be observed at the sum of the two electron Zeeman frequencies, i.e., at twice
the electron Zeeman frequency of the nitroxide radical. Since cw EPR experiments are
performed at constant mw frequency and variable field, the double-quantum transition is
observed at half the field of the usual single-quantum spectrum.
Figure 10.45: Energy level scheme for a system consisting of two electron spins S
1
= 1/2 and S
2
= 1/2
with assignement of the terms of the dipolar alphabet and of zero-quantum (ZQ), single-quantum (SQ),
and doubl;e-quantum (DQ) transitions.
The intensity of the half-field transition depends on the ratio between the dipole-dipole
coupling and the electron Zeeman interaction. The distance can thus be determined from the
integral intensities V(DQ) of the double-quantum (DQ) transition and V(SQ) the four single
quantum (SQ) transitions.
1
Numerical computations provide the approximate relation
2
[10.85]
that is valid up to X band frequencies. The approach neglects anisotropic contributions of the
exchange coupling, as is appropriate for distances larger than about 6 . For distances larger
1
S. S. Eaton, G. R. Eaton, J. Am. Chem. Soc. 1982, 104, 5002-5003. Measurement of Spin-Spin Distances
friom the Intensity of the EPR Half-Field Transition.
2
R. E. Coffman, A. Pezeshk, J. Magn. Reson. 1986, 70, 21-33. Analytical Considerations of Eatons For-
mula for the Interpsin Distance between Unpaired Electrons in ESR.
oo ' , || ' , E

oo ' , || ' ,
|bb
|ba
|ab
|aa
B
^
E F
^ ^
C,D
C,D
C,D
C,D
^
^
^
^
^
^
^
^ SQ
SQ
SQ
SQ
DQ
ZQ
V DQ ( )
V SQ ( )
------------------ 19.5 10.9Ag + ( )
9.1 GHz
v
mw
--------------------
\ .
| |
2

6
r
6
------ =
245
than about 12 the intensity of the half-field transition at X-band frequencies is too weak for
this kind of analysis.
10.6.2 DEER
At distances longer than 18... 20 the dipole-dipole coupling needs to be separated
from other interactions that lead to broadening of EPR lines. The experimentally most robust
technique for such separation is the double electron electron resonance (DEER) experiment,
which is also known als pulse electron electron double resonance (PELDOR). By employing
two mw frequencies this experiment excites each of the two coupled spins separately. It is thus
akin to a heteronuclear rather than a homonuclear NMR experiment. The separate excitation of
two electron spins is possible even if they both belong to nitroxide radicals, as the excitation
bandwidth of mw pulses is significantly smaller than the width of the nitroxide spectrum. The
two frequencies thus select different orientations or different spin states of the
14
N nucleus in
the two radicals (Fig. 10.46).
Figure 10.46: Selective excitation of two radicals by observer and pump pulses in a doubly nitroxide
labeled macromolecule. The radicals have different orientations with respect to the external magnetic
field or different
14
N nuclear spin states and thus different resonance frequncies.
The subsequence for the observer spin A at frequency v
1
is a refocused echo sequence
(Fig. 10.47). It consists of a part (t/2)-t
1
-(t)-t
1
that creates a first echo and a part t
2
-(t)-t
2
that
refocuses the signal once again and creates a second echo. This second echo is observed. As
the observer t pulses excite only the A spin, they refocus all interactions that are linear in
operators of this spin. These are the electron Zeeman interaction, the hyperfine interaction of
the A spin, and also the dipole-dipole interaction between the A spin and the pumped B spin.
pump ( ) n
2
observer ( ) n
1
B
0
N N O
O
.
.
B
0
excitation bands
schematic spectra
A
B
246

Only the dipole-dipole interaction is reintroduced by the pump t pulse at frequency v
2
,
which exclusively excites the B spin. This pump pulse inverts the state of the B spin and thus
leads to a change of the resonance frequency of the observer spin A by the dipole-dipole
splitting d(u) given in Eq. [10.31]. This frequency change is induced at a variable delay t with
respect to the first observer echo, while delays t
1
and t
2
are fixed. The echo signal is thus
modulated with e
dd
as a function of time t. From e
dd
the distance can be determined using Eq.
[10.30] or [10.32]. The modulation is not damped by relaxation, as the total duration of the
experiment is constant. Nevertheless distance resolution and the upper distance limit depend
on electron spin relaxation, as the maximum observation time t
max
of the dipolar modulation
cannot be much longer than T
2
. Therefore, the experiment is performed at temperatures of
about 50 K, where transversal electron spin relaxation of nitroxides asymptotically approaches
its low-temperature maximum. At this temperature transversal relaxation is mainly driven by
fluctuating hyperfine fields from weakly coupled protons. A decrease of proton concentration
by deuterium exchange extends the upper distance limit and improves resolution.
Figure 10.47: Pulse sequence of the DEER experiment consisting of the refocused echo subsequence at
frequency v
1
that excites exclusively observer spins A and a pump pulse at frequency v
2
that excites
exclusively pumped spins B. Delays t
1
and t
2
are fixed, while delay t is varied. The echo amplitude
oscillates with the dipolar splitting d(u) as a function of delay t.
As the pump pulse has limited excitation bandwidth and inverts only a fraction of the
B spins, only a fraction of the echo intensity is modulated. The remaining fraction 1- of the
echo amplitude is independent of t for an isolated pair. The DEER signal F(t,u) for an isolated
spin pair is thus given by
. [10.86]
The form factor F(t) for a macroscopically disordered sample is obtained by powder averaging
p/2
p
p
p
t
t
t 1
2
n
1
n
2
F t u . ( ) F 0 ( ) 1 u ( ) 1 d u ( )t ( ) cos | | { } =
247
. [10.87]
Figure 10.48: DEER distance measurement on a rigid biradical. A) Structure of the biradical. B)
Normalized echo amplitude as a function of delay t. The read line is a fit of the background function
B(t) according to Eq. [10.88]. C) Form factor F(t) obtained by dividing the normalized echo amplitude
by the background function. The red line is the theoretical form factor corresponding to the extracted
distance distribution P(r). D) Dipolar spectrum (Pake pattern) obtained by subtracting the constant part
from the form factor F(t), multiplication with a Hamming window, zero filling, and Fourier
transformation. The red line is the theoretical spectrum corresponding to the extracted distance
distribution P(r). E) Distance distribution P(r) obtained by Tikhonov regularization.
Interaction of the A spin with spins in neighboring molecules modifies the signal, as a
fraction of theses spins is also inverted by the pump pulse. If the neighboring spins are
homogeneously distributed in space, the background factor B(t) assumes the form
F t ( ) V t u . ( ) u sin u d
0
t 2

=
N N
O
O
O
O
N N O
(CH2)5CH3
H
3
C(H
2
C)
5
(CH2)5CH3
H
3
C(H
2
C)
5
(CH2)5OCH3
H
3
CO(H
2
C)
5
O
0 5 10
0.6
0.7
0.8
0.9
1
t (s)
0 5 10
0.8
0.9
1
t (s)
3 4 5 6
r (nm)
-1 0 1
n (MHz)
A
B
D
C
E
background
Tikhonov regularization
F
o
u
r
i
e
r
correction
t
r
a
n
s
f
o
r
m
a
t
i
o
n
V
t
V
(
)
/
(
0
)
P
r
(
)
I
(
)
n
n
dd
l
248

, [10.88]
where the average modulation depth is the fraction of spins excited by the pump pulse, g is
an average g value, and c is the total concentration of spins. The total DEER signal
[10.89]
for a well-defined distance between the spins has an appearance as shown in Fig. 10.48B.
The form factor for the isolated pair can thus be obtained by fitting the background
function and dividing the DEER signal V(t) by this function (Fig. 10.48B). The dipolar
spectrum is computed by Fourier transformation after removing the constant component 1- of
the form factor (Fig. 10.48C). From the singularity of the Pake patern, v
dd
can be determined
and the distance calculated according to Eq. [10.32].
Due to flexibility of the macromolecule and conformational degerees of freedom of the
label, the spin-spin distance is not sharply defined but rather distributed. Extraction of this
distribution of distances P(r) from the form factor F(t) is an ill-posed problem. In ill-posed
problems small errors in the input data F(t) may result in large errors of the output data P(r).
Errors in the input data come from noise or incomplete background correction and are
unavoidable. Therefore the solution has to be stabilized by additional constraints. Constraints
can be derived from the properties of the distribution to be non-negative (P(r)>0 for all
distances r) and smooth. Smoothness corresponds to a small square norm n of the second
derivative
. [10.90]
Furthermore, the mean square deviation p of the simulated form factor S(t) from the
experimental form factor F(t),
, [10.91]
has to be small (goodness of the fit). The relative weight of these two criteria for a good
solution of the problem is experessed by a regularization parameter o. The best solution for the
distance distribution is obtained by minimizing
B t ( )
2tg
2
u
B
2
u
0
N
A
9 3h
----------------------------------ct
\ .
| |
exp =
V t ( ) F t ( )B t ( ) =
n
r
2
d
d
P r ( )
2
=
p S t ( ) F t ( )
2
=
249
[10.92]
This solution can be directly computed by matrix algebra with the Tikhonov regularization
algorithm.
The result of Tikhonov regularization depends on the choice of the regularization
parameter o. A good compromise between undersmoothing (too small o) and oversmoothing
(too large o) can be found by plotting log n(o) vs. log p(o). Because of its typical shape this
plot is termed L curve (Fig. 10.49). In the steep branch of the curve at small o. an increase in o
leads to a strong decrease in the square norm (much smoother distribution), but to only a small
increase in the mean square deviation of the simulated from the experimental data. In the flat
branch at large o, a further increase of o causes a strong increase in the mean square deviation
(deterioration of the fit), but to only a small improvement in smoothness. The best compromise
corresponds to the corner of the L (arrow in Fig. 10.49).
Figure 10.49: L curve for the distance distribution of the biradical in Fig. 10.48. The optimum
regularization parameter o= 0.1 corresponds to the corner of the L (red). The distance distribution
obtained with this regularization parameter is shown in Fig. 10.48E.
Determination of the distance distribution rather than only the mean distance is
particularly important for systems that simultaneously attain two or more conformational
states. This situation is encountered in studies of protein function or folding.
G
o
p on + =
-5 -4.5 -4
-20
-16
-12
-8
l
o
g
h
log r
a= 10
-5
a= 10
-1
a= 10
3
250

10.7 Technical considerations
10.7.1 The cw EPR experiment
The general setup of an EPR spectrometer is similar to the one of an NMR
spectrometer discussed in Chapter 2.5 (Fig. 10.50). The mw source in modern spectrometers is
a Gunn diode whose frequency can be tuned over a range of typically 1 GHz. The NMR coil
and capacitance are substituted by a cavity resonator (Fig. 10.51A) or, for special experiments,
a loop-gap resonator. The dimensions of cavity resonators are comparable to the wavelength of
the mw (Table 10.1). The standing wave in the resonator has spatial regions with a large
magnetic and a small electric component of the alternating field (Fig. 10.51B). This is where
the sample has to be placed, as resonant absorption of the magnetic component provides the
signal, while non-resonant absorption of the electric component by the electric dipoles of
molecules in the sample leads to mw losses and sample heating. Therefore sample size must be
much smaller than the wavelength. Typical outer diameters of sample tubes for cw EPR are 4
mm at X band, 1.6 mm at Q band, and 0.9 mm at W band. Samples that contain highly polar
solvents, such as water, have high dielectric losses and thus need to be confined to a region
where alternating electric fields are very small. They are thus measured in capillaries or flat
cells. Typical sample amounts are 150 mg/150 ul at X band and 1 mg/1 ul at W band for non-
lossy samples and by about a factor 2 to 5 less for aqueuos samples. It is possible to routinely
detect micromolar concentrations with a microliter of sample at X band, corresponding to 1
pmol of or paramagnetic centers.
Figure 10.50: Schematic drawing of a cw EPR spectrometer.
6
11
10
microwave
source
reference
arm
bias
attenuator
resonator
magnet
modulation
coils
1
2
3
f
m.w.
diode
PSD
modulation
generator
S
i
g
n
a
l
circulator
phase
251
The mw from the source (power up to P
mw
= 200 mW at X band) is attenuated to a
level that does not cause saturation of the electron spin transitions (see Section 2.7.2). With
typical attenuations between 20 and 40 dB the power incident on the sample is about 2 mW to
20 uW. This power reaches the cavity resonator through a circulator, so that no power can
directly pass on to the mw diode that functions as the detector (Fig. 10.50). The mw is
transmitted via a waveguide that is coupled to the resonator by an iris with a tuning screw (Fig.
10.51). For best sensitivity the spectrometer has to be tuned and matched.
Figure 10.51: Cavity resonator used in cw EPR experiments. A) Rectangular cavity with iris tuner. The
mw is fed from the left front side by a rectangular waveguide. B) Distribution of the mw field. Solid
lines with arrows marked B
1
correspopnd to the magnetic field component, while dots and crosses
correspond to the electric field component.The central plane of the resonator is a nodal plane of the
electric field.
Tuning means that the frequency of the Gunn diode is adjusted to the resonance
frequency of the cavity resonator. For that the mw absorption of the cavity is measured as a
function of frequency and displayed as a tuning picture (Fig. 10.52). The dip is moved to the
center of the picture by adjusting the center frequency of the sweep.
Maximum sensitivity is achieved if the impedance R
0
of the transmission line (50 O) is
matched to the impedance R of the cavity resonator. Due to the non-resonant mw losses, R
depends on the dielectric properties of the sample. Tuning and matching thus have to be
repeated for each sample. A matched cavity does not reflect any mw power if the electron
spins are off resonance (Fig. 10.52). This means that zero mw power is transmitted through
B
1
B
1
A B
252

ports 2 and 3 of the circulator towards the detecting mw diode (Fig. 10.50). Matching is
achieved by adjusting the iris tuning screw. This changes the coupling n between the
transmission line and the resonator until
, [10.93]
a condition that is termed critical coupling. The loaded quality factor, which characterizes the
resonant enhancement of the mw field in the cavity, is then given by
, [10.94]
where L is the inductivity of the cavity. The amplitude of the magnetic field component B
1
becomes
, [10.95]
where V
c
is the effective volume of the resonator, which scales roughly as . For the
same Q
L
, less mw power P
mw
is thus required at higher frequencies to achieve the same
amplitude of the alternating magnetic field. More power is needed if the sample is lossy (large
R), corresponding to low Q
L
. .
Figure 10.52: Idealized tuning picture of a matched (solid line) cavity resonator with a loaded quality
factor Q
L
= 10000 at X band frequencies. The dashed line corresponds to resonant mw absorption by
the electron spins.The EPR signal AV
refl
is due to the power reflected in this situation
The bandwidth Av of the resonator (Fig. 10.52) is given by
R
0
n
2
R =
Q
L
2tv
resonator
L
R
0
n
2
R +
--------------------------------- =
B
1
2Q
L
P
mw
u
0
V
c
v
resonator
----------------------------------- =
v
resonator
3
-2 -1 0 1 2
n-n
resonator
(MHz)
Dn
no reflection
total reflection
-3 dB
s
i
g
n
a
l
v
o
l
t
a
g
e
a
f
t
e
r
m
.
w
.
d
i
o
d
e
DV
refl
253
. [10.96]
In cw EPR the bandwith of a high-quality cavity resonator is always sufficient. For
pulsed EPR, bandwidths of 30 to 200 MHz are required. This is achieved by intentionally
spoiling resonator quality (increasing R) or by overcoupling (increasing n beyond critical
coupling).
With the spectrometer tuned and matched, the measurement is performed by keeping
the mw frequency constant and sweeping the magnetic field through resonance of the electron
spins. On resonance, the spin systems absorbs mw energy and dissipates the energy through
longitudinal relaxation. This corresponds to an increase in cavity impedance R and thus to a
violation of the critical coupling condition, Eq. [10.93]. The cavity is no longer matched and
mw power is reflected (Fig. 10.52). This reflected power is transmitted through ports 2 and 3
of the circulator to the detecting mw diode (Fig. 10.50). The change in the voltage incident at
the mw diode is given by
, [10.97]
where ;(B
0
) is the field-dependent imaginary part of magnetic susceptibility arising from the
electron spins, n is the filling factor of the resonator and C is an apparative constant. The
function ;(B
0
) is the EPR absorption spectrum. The filling factor is the ratio of the integrals
of the mw magnetic field amplitude over the sample and the whole resonator,
. [10.98]
Sensitivity of the cw EPR experiment can thus be increased by optimizing either the quality
factor Q
L
(cavity resonators) or the filling factor n (loop-gap resonators, dielectric ring
resonators). The first strategy works better for samples with low dielectric losses that are
available in sufficient quantities (100 mg). The second strategy is preferable for lossy samples
or small sample quantities. Dielectric resonators combine high Q
L
and high n. They are only
used for special applications or pulse EPR as the dielectric rings used for concentrating the mw
field have background signals.
Av
v
resonator
Q
L
---------------------- =
AV
refl
C; B
0
( )nQ
L
=
n
B
1
V d
sample

B
1
V d
resonator

--------------------------------- =
254

The reflected mw power is detected by a mw diode, which can manufactured with a
low intrinsic noise figure. However, mw diodes have two drawbacks. First, they are not
sensitive at very low incident powers (Fig. 10.53). Second, they detect mw power over a very
broad frequency range, thus also collecting thermal noise from this whole frequency range.
These problems require two additions to the spectrometer.
To achieve best sensitivity and linearity of the output signal with respect to incident
voltage, the diode has to be biased to its operating point (Fig. 10.53). This is done by
transmitting a small fraction of the mw power directly from the source to the diode via the
reference arm (Fig. 10.50). The operating point is attained by adjusting the bias attenuator. The
output current with the sample off resonance should correspond to the horizontal dotted line in
Fig. 10.53 (typically 200 uA). The mw transmitted through the reference arms must have the
same phase as the mw reflected from the resonator to interfere constructively at the diode
input. This is achieved by adjusting the mw phase in the reference arm.
Figure 10.53: Characteristic curve of an mw diode for cw EPR detection (solid line). Highest
sensitivity and linear behavior is obtained near the operating point (full circle). The diode is damaged at
too high incident power (full diamond). In modern spectrometers, the diode is protected from damage
by limiting the input power. The characteristic curve of a limiter-protected diode is shown as a dashed
line.
The detection band can be narrowed and thus much of the noise excluded by imposing
a modulation on the signal. This can be done most easily by modulating the external magnetic
field at a low frequency that can pass the mw. diode (typically 100 kHz). Such modulation
leads to an oscillation of the reflected mw with the same frequency (Fig. 10.4). For sufficiently
low field modulation amplitudes AB
0
the amplitude of the voltage oscillation is proportional to
the derivative , i.e., the derivative of the absorption spectrum. This amplitude is
incident voltage
o
u
t
p
u
t
c
u
r
r
e
n
t
operating
point
d; dB
0

255
measured by a phase-sensitive detector (PSD). As a result cw EPR spectra are derivative
absorption spectra.
With increasing modulation amplitude the signal increases, while noise remains the
same. This applies until the modulation amplitude reaches the peak-to-peak linewidth AB
pp
.
With this modulation amplitude detection is most sensitive. However, if the modulation
amplitude exceeds AB
pp
/3, the line is artificially broadened. Therefore, the modulation
amplitude is adjusted to about one third of the width of the most narrow line in the spectrum. In
rare cases natural linewidths can be smaller than 100 kHz, corresponding to 36 mG or 3.6 uT
on a magnetic field axis. In this case the modulation frequency has to be decreased to avoid
line broadening. Finally, saturation of the electron spin transitions (see Section 2.7.2) may also
cause line broadening. Such power broadening is avoided in the linear regime where the signal
amplitude increases with the square root of the mw power, . In this regime the
amplitude increases by a factor of two when decreasing attenuation by 6 dB
( ).
Figure 10.54: Schematic drawing of a pulse EPR spectrometer.
10.7.2 Pulsed EPR experiments
In a pulsed EPR spectrometer the pathways for excitation power, signal and reference
mw are similar to the ones in a cw spectrometer (Fig. 10.54). The mw pulses are created in an
mw pulse forming unit (MPFU), where the mw power is divided into several channels. In the
AV P
mw

10
0.6
4 4 . 2 = =
m.w.
source
2 m.w.
source
nd
r.f.
source
r.f.
PFU
reference arm
main
attenuator
magnet
1
2
3
f
MPFU
*
*
MPFU m.w. pulse forming unit
**
S protection switch or power limiter
S
**
power
amplifier
receiver
amplifier
m
i
x
e
r
v
i
d
e
o
a
m
p
l
i
f
i
e
r
r.f. amplifier resonator video signal
(MHz range)
r.f. coils
E
L
D
O
R
E
N
D
O
R
phase shifter
circulator
256

most flexible setup each channel has its own attenuator, phase shifter, and PIN diode switch
(Fig. 10.55A). This allows for independent adjustment of power, phase, and timing of the
pulses. The PIN diode switches have rise and fall times of about 3-4 ns, so that a pulse with a
length of 32 ns is not quite rectangular (Fig. 10.55B). The power from all channels is
recombined at the output of the MPFU. Modern commercial spectrometers have one preset
four-channel MPFU with fixed quadrature phases (+x, +y, -x, -y). The power of the four
channels cannot be adjusted independently. This MPFU provides best performance in phase
cycling and ease in experiment setup. For full flexibility, additional MPFUs with adjustable
phase and amplitude in each channel have to be installed
Figure 10.55: MW pulse formation. A) MW pulse forming unit (MPFU) with four channels whose
amplitudes and phases can be adjusted independently. B) Typical pulse shape in EPR with the rise and
fall times of the pulse-forming switch being comparable to the pulse length.
Much larger B
1
fields are required for pulse EPR than for cw EPR. Therefore, the mw
excitation power is amplified in a traveling wave tube ( kW) or solid-state power
amplifier ( W). A precision attenuator after this amplifier allows for adjusting pulse
power. The pulses enter the resonator via the circulator. Critical coupling of the resonator is
not required in pulsed EPR, since excitation and detection are separated in time. In fact,
resonators are usually overcoupled on purpose to increase bandwidth (see Eqs. [10.94,10.96]).
For instance, a pulse with a length t
p
= 12 ns has an excitation bandwidth of about 1/t
p
= 83
MHz and thus requires Q
L
< 115 at a frequency of 9.6 GHz. Such overcoupling leads to
substantial power reflection. Reflected mw power in the range of tens to hundreds of Watt
would destroy the sensitive receiver amplifier. Therefeore, the amplifier is protected by a
switch or a power limiter.
During detection the protection switch is open. The signal is amplified by about 20 dB
by the mw receiver amplifier. This amplifier has a very low noise figure. The signal is then fed
to a mixer, where a video signal at the difference frequency between the reference mw and the
MPFU
S
+ x
S
+ y
S
- x
S
- y
j
j
j
j
attn. phase PIN diode
t = 0
t
p
A B
32 ns
P
mw
1 ~
P
mw
10 ~
257
signal mw is generated. This frequency subtraction corresponds to detection in the rotating
frame. Sign information on the difference frequency is lost, unless two mixers with a reference
phase difference of are used. Such quadrature detection with a doubly-balanced mixer is
optionally available in modern EPR spectrometers. It should be used only when necessary, as
the splitting of the signal to two mixers decreases signal-to-noise ratio compared to single-
channel detection.
The video signal is passed through a low-pass filter, amplified, and detected by a fast
digitizer with a typical time resolution between 1 and 8 ns. Modern digitizers allow for signal
accumulation at repetiton rates up to 1 MHz. In echo experiments, the digitized signal is
integrated over a certain time range. This improves signal-to-noise ratio. The width of the
integration gate puts an upper limit on the frequencies that can be detected in the signal.
Optimum sensitivity is achieved by matching detection bandwidth to excitation bandwidth.
This corresponds to an integration gate that is approximately as wide as the full width at half
height of the echo, which is in turn given by the length of the longest excitation pulse (window
with length t
intg,SN
in Fig. 10.56). For recording field-swept echo-detected EPR spectra the
detection bandwidth should be much smaller than the width of the most narrow peaks in the
absorption spectrum. This is achieved by integrating over the whole echo. Typical widths for
the integration gate in this mode of operation (t
intg,res
in Fig. 10.56) are 100 to 200 ns.
Figure 10.56: Signal trace in a two-pulse echo experiment starting immediately after the falling edge of
the t pulse (simulation for an infinetely broad inhomogeneous line). Pulse lengths of 16 and 32 ns were
assumed for the t/2 and t pulse, respectively.
90
0 100 200 300 400
t (ns)
FID
echo
t
d
t'
t
intg,SN
t
intg,res
s
i
g
n
a
l
v
o
l
t
a
g
e
(
a
.
u
.
)
t
p
258

The theoretically expected signal trace after a two-pulse sequence with an interpulse
delay t measured between the falling edge of the t/2 pulse and the rising edge of the t pulse is
shown in Fig. 10.56. The simulation assumes that the EPR line is inhomogeneously broadened
and is much broader than the excitation bandwidth, as is often the case. For such a line, the FID
length t
FID
is determined by the length of the t pulse. The echo appears at a time slightly
longer than t, as the effective evolution time is approximately the delay between the centers of
the two mw pulses. In practice, only the echo but not the FID can be detected, as the signal
with a power of a few microwatts is superimposed by stronger residual power from the high-
power pulses within dead time t
d
. For a residual power of 100 W to decay to the level of 1 uW
a time ln(10
8
)t
ring
has to pass, where t
ring
is the ringing time of the system. This time cannot be
shorter than the ring down time of the resonator t
ring,res
= Q
L
/(tv
mw
). For Q
L
= 100 at 9.6 GHz
this corresponds to a dead time of approximately 60 ns. Any reflection of power in the mw
bridge increases t
ring
and proportionally t
d
.
The decrease in Q
L
that allows for a larger excitation bandwidth leads to a loss in
sensitivity compared to cw EPR. This loss is only partially compensated by the contribution of
more spin packets to the signal. Furthermore, according to Eq. [10.95] this decrease also leads
to a smaller mw field amplitude B
1
at given power P
mw
. These unwanted effects can be
compensated by a different resonator design than in cw EPR. While cw EPR resonators are
optimized by maximizing Q
L
, pulse EPR resonators are optimized for high filling factors n
(Eq. [10.98]) and for effective volumes V
c
that match available sample volume as closely as
possible. Both criteria require a confinement of the magnetic component of the mw field to
dimensions that are much smaller than the wavelength in air. There exists the additional
constraint that the electric field component in the sample should be as small as possible to
avoid sample heating.
The dimensions of an mw resonators can be reduced by filling it with a material with
much larger dielectric constant c than the one of air (c= 1). The mw wavelength is inversely
proportional to . Furthermore, if a hole is drilled in the dielectric medium, the electric
component is mainly condined in the dielectric ring and the magnetic component in empty
space. Such a dielectric ring (Fig. 10.57A) within a cylindical cavity resonator combines a
large filling factor n, small effective volume, and high loaded quality factor Q
L
at critical
coupling. When critically coupled it allows for cw EPR experiments with good sensitivity and
c
259
when overcoupled for pulse EPR experiments with good power conversion. Compared to an
optimized cavity resonator without dielectric ring it has somewhat lower cw EPR sensitivity.
For some cw EPR applications and pulse EPR at very low temperatures it is not suitable, as
dielectric materials generally have some background signal due to transition metal impurities.
Usually sapphire ( ) is employed.
Figure 10.57: Resonator designs used for pulsed EPR. A) Dielectric ring resonator, consisting of a ring
of material with a high dielectric constant c that is placed inside an mw shield or outer cylindrical
resonator. The sample is placed in the center hole. B) Loop-gap resonator consisting of a metal ring
with a vertical slit. The sample is placed in the center hole.
More flexibility in scaling of the critical volume is achieved with lumped circuit
resonators, which can be pictured as LC resonance circuits with the inductivity L and the
capacitance C in the same place. More strictly defined the travel time of the electric signal
between the elements of a lumped circuit is negligible. The most simple lumped circuit
resonator is the loop-gap resonator, which is a massive metal ring with a slit that has a constant
width (Fig. 10.57B). The slit (gap) can be considered as the capacitance C while the metal
piece is a one-turn coil, i.e., the inductivity L. Accordingly, the electric component of the mw
field is concentrated across the gap, while the magnetic component is particularly strong inside
the ring (loop), where the sample is placed. The dimension of a loop-gap resonator can be
adjusted without changing its frequency, as L and C can be varied independently by changing
the radius of the loop and the width of the gap, respectively.
An antenna is used to couple mw into such a loop-gap resonator. The coupling n can be
changed by shifting the antenna vertically (along the B
1
direction) with respect to the
resonator. Such antenna coupling can also be employed for the dielectric ring resonator.
c 10 ~
B
1
B
0
B
1
gap
loop
A B
260