EPR at Work
JAMES S. HYDE, SANDRA S. EATON, GARETH R. EATON
1
National Biomedical EPR Center, Department of Biophysics, Medical College of Wisconsin,
Milwaukee, Wisconsin, 53226
2
Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado 80208
INTRODUCTION
The background and early history of the famous series
EPR at Work was sketched in the chapter by J.S.
Hyde, “EPR at Varian: 1954 –1974,” in Foundations
of Modern EPR (1). The series began in 1957 with a
description of the hyperfine structure of free radicals,
illustrated with a spectrum of Fremy’s salt. The early
articles were written by Larry Piette and Richard H.
Sands. Of the 47 articles, 13 were written by J.S.
Hyde. The early EPR at Work articles were written in
parallel with longer, tutorial articles in the Technical
Information Bulletin. After the EPR at Work series,
there were a few similar reports with the title EPR in
the World of Biochemistry. Varian produced these
reports as a marketing tool, published as advertise-
ments in major scientific journals, but they were solid
scientific reports that in some cases served as “pre-
liminary communications” for later full articles. For
scientists of the time, the EPR at Work, and the
similar NMR at Work, series served to alert them to
the wonderful things that could be done with the new
magnetic resonance tools becoming commercially
available.
Several of the “At Work” publications revisit sim-
ilar topics of great importance. Many of the EPR at
Work articles were followed by research articles in
journals such as the Journal of Chemical Physics. The
EPR at Work series stimulated many scientists to
include EPR as a tool in their research. Some of the
spectroscopy reported remains educational and in
Received 11 November 2005; accepted 11 November
2005
Correspondence to: James S. Hyde; E-mail: jshyde@mcw.edu
Original Varian tutorial articles are reproduced, with commentary,
by James S. Hyde, Sandra S. Eaton, and Gareth R. Eaton.
Concepts in Magnetic Resonance Part A, Vol. 28A(1) 1–100 (2006)
Published online in Wiley InterScience (www.interscience.wiley.
com). DOI 10.1002/cmr.a.20047
© 2006 Wiley Periodicals, Inc.
1
some cases a benchmark of EPR. We reproduce them
on the following pages (with the permission of Var-
ian) with the hope that a new generation of scientists
will be stimulated by them. We add our perspective
on these early reports with brief comments and mod-
ern references. It is immediately striking that the
topics remain current; in fact, many remain challenges
for further research.
EPR at Work No. 1: Hyperfine Structure in Free
Radicals
EPR at Work No. 2: Magnetic Molecules in Oxi-
dation-Reduction Equilibria
EPR at Work No. 3: Magnetic Intermediates in
Oxidation-Reduction Equilibria
EPR at Work No. 4: Chemical Kinetics of Free
Radical Reactions
EPR at Work No. 5: Vanadium Ions in Crude Oil
EPR at Work No. 6: Paramagnetic Ions in Micror-
ganisms
EPR at Work No. 7: Paramagnetic Resonance Ab-
sorption in Vanadyl Chelate
EPR at Work No. 8: Damage from Freeze Drying
in Whole Blood
EPR at Work No. 9: Free Radical Intermediates in
Enzyme-Substrate Reactions
EPR at Work No. 10: Free Radical and Paramag-
netic Ion Kinetics in Enzyme-Substrate Reactions
EPR at Work No. 11: Free Radical Intermediates in
the Action of Oxidation Inhibitors
EPR at Work No. 12: Free Radical Formation in
Irradiated Polymers
EPR at Work No. 13: A Free Radical Condensation
of Biological Interest
EPR at Work No. 14: Disappearing Free Radicals
EPR at Work No. 15: Free Radicals by Irradiation
EPR at Work No. 16: Irradiation Studies at Very
Low Temperatures
EPR at Work No. 17: Forbidden Transitions in a
Vanadyl Chelate
2 HYDE, EATON, AND EATON
EPR at Work No. 18: High Sensitivity EPR as a
Tool in Free Radical Research
EPR at Work No. 19: High Sensitivity EPR as a
Tool in Free Radical Research
EPR at Work No. 20: High Sensitivity EPR as a
Tool in Solid State Research
EPR at Work No. 21: Hyperfine Structure in Or-
ganic Free Radicals by EPR
EPR at Work No. 22: Hyperfine Structure in Or-
ganic Free Radicals by EPR
EPR at Work No. 23: EPR High Sensitivity in
Aqueous Solutions
EPR at Work No. 24: Study of a Chemical Reac-
tion Mechanism with EPR
EPR at Work No. 25: Radiation Damage Studies
with High Sensitivity EPR
EPR at Work No. 26: EPR of Triplet States
EPR at Work No. 27: Second Derivative Display in
EPR
EPR at Work No. 28: Precision Measurements in
EPR
EPR at Work No. 29: Precise EPR Hyperfine Mea-
surements
EPR at Work No. 30: EPR of Triplet States
EPR at Work No. 31: Large Line Separations in
EPR
EPR at Work No. 32: High Resolution EPR
EPR at Work No. 33: High Sensitivity EPR in
Photosynthesis
EPR at Work No. 34: Ferro- and Antiferromag-
netism Studies with EPR
EPR at Work No. 35: EPR at 35 Gc
EPR at Work No. 36: EPR at 35 Gc
EPR at Work No. 37: EPR Hyperfine Structure
from Isotopes in Low Abundance
EPR at Work No. 38: EPR at 35 GHz
EPR at Work No. 39: Spin Labels for Biomol-
ecules
EPR at Work No. 40: Liquid Crystal Alignment
Studied by EPR
EPR at Work No. 41: Liquid Crystals as Solvents
in EPR
EPR at Work No. 42: Prognostic Studies by EPR
EPR at Work No. 43: Molecular Structure Deter-
minations by EPR
EPR at Work No. 44: EPR Probing of Antibodies
EPR at Work No. 45: EPR Studies of Membrane
Structure
EPR at Work No. 46: Precision g Values
EPR at Work No. 47: EPR of Radiation Induced
Radicals
EPR in the World of Biochemistry–Identification
of Free Radicals
Concepts in Magnetic Resonance Part A (Bridging Education and Research) DOI 10.1002/cmr.a
EPR in the World of Biochemistry–Stoichiometry
of a Free Radical Reaction
EPR in the World of Biochemistry–Excited Triplet
States in Aromatic Amino Acids
EPR in the World of Biochemistry–EPR Monitor-
ing of Drug Levels in Blood Sera
EPR in the World of Biochemistry–Monitoring
Ascorbate Levels in Blood Sera
In addition to these series, Varian produced a tech-
nical information bulletin that contained several arti-
cles about EPR. These will be referred to as TIB
followed by the date. The EPR at Work articles will
be referred to by number, i.e., no. 3, and so on.
1957: EPR Spectroscopy, a Companion to NMR
1959: Improved EPR Techniques for Free Radical
Detection in Photochemistry
1961: Application of EPR to Biological Studies
1962: Operational Considerations with the V-4532
Dual Sample Cavity
1965: Signal Amplitudes in Electron Paramagnetic
Resonance
1965/66: EPR at 35 GHz
1966, Spring: Some EPR Properties of the Rare
Earths: Part I
1966, Fall: Some EPR Properties of the Rare
Earths: Part II
EPR AND ITS APPLICATIONS
TIB 1957 provided an introduction to EPR. It ex-
plained how an EPR spectrometer works, and it illus-
trated applications to spin delocalization in semiqui-
none radicals, spin exchange as a function of
concentration in solutions of DPPH, and metal nu-
clear hyperfine in aqueous solutions of Mn2⫹. Many
modern textbooks introduce EPR in much the same
way, adding information about hyperfine rather like
that in no. 1. An author of this current article wrote an
introduction to EPR with a focus on biochemical
applications (2) and a review of instrumental aspects
of EPR in a microwave engineering volume (3). The
other authors wrote an introduction to EPR that en-
compasses CW and time-domain methods (4). More
extensive treatments, at a level appropriate to readers
with some familiarity with physical chemistry, are in
the textbooks by Atherton (5) and by Weil, Bolton,
and Wertz (6). The most comprehensive discussion of
basics of EPR instrumentation and methodology is the
treatise by Poole (7, 8). Much practical “string and
sealing wax” information about EPR is in the book by
Alger (9).

The EPR at Work nos. 18, 19, 20, 23, and 33
highlighted the signal-to-noise advantages of using
100 kHz magnetic field modulation.
CLASSES OF SAMPLES
The frequently cited “eight classes of samples” (yes or
no for saturable, limited, or dielectric loss) was pre-
sented by Hyde in TIB 1965. The discussion of di-
electric properties of the sample was extended by
Dalal et al. (10) and in a recent series of articles from
the Hyde lab (11–13). For extensive discussion of
frequency dependence and sensitivity, see Rinard et
al. (14 –16).
HYPERFINE STRUCTURE
EPR at Work nos. 1, 21, 22, 29, 32, 37, and 43 all
discuss hyperfine structure, and many others use hy-
perfine patterns in the analysis of the spectra. Most
books that discuss EPR describe hyperfine patterns in
some detail. Weil, Bolton, and Wertz (6) and Ather-
ton (5) provide solid background on hyperfine, in-
cluding second-order effects. For organic radicals, the
book by Gerson and Huber (17) gives an up-to-date
summary. Note the discussion of second-order shifts
in no. 29, a topic of increasing importance as biolog-
ical studies use microwave/RF frequencies below X-
band. Number 37 uses higher gain to show the hyper-
fine splitting from low-abundance nuclei in Fremy’s
salt, which was evident only to the practiced eye in
the spectrum of this radical shown in no. 1. Detailed
studies of spin exchange of Fremy’s salt in solution
were reported by Jones (18). Relaxation times by line
width and CW saturation were measured by Rataiczak
and Jones (19). Because there is no unresolved hy-
perfine structure for Fremy’s salt, the low-concentra-
tion line width yields T2 and then the power saturation
curve yields T1. The spectrum of tetracene cation
radical, formed by dissolving tetracene in H2SO4, was
published by Hyde and Brown (20) (listed as “to be
published” in no. 21). Note the discussion in this
article of the difficulties of achieving the 1-ppm field
stability needed to record the narrow lines (35 mG) of
this radical. This article also used a dual cavity and
applied the perturbing spheres method for measuring
B1 in the cavity. Number 22 showed the hyperfine
splittings in a radical generated electrochemically,
and highlighted the method developed by Maki and
Geske for generating radicals electrochemically in the
EPR cavity. This has become a major method of
creating radicals (21–25).
Concepts in Magnetic Resonance Part A (Bridging Education and Research) DOI 10.1002/cmr.a
EPR AT WORK 3
The hyperfine splittings discussed in the EPR at
Work series can be used to identify free radicals
produced during enzymatic oxidation of several sub-
strates. Reductic acid, with four equivalent protons,
yields a radical with a five-line spectrum, ascorbate
has major coupling to a single proton so there are two
lines in the spectrum, and the radical generated from
dihydroxyfumaric acid, having no protons, yields a
single-line EPR spectrum.
Effects of H versus D and ring substitution on the
hyperfine patterns in a series of naphthoquinones (no.
43) reveal, for example, splitting by the H of OH
groups. Among the examples is one in which coupling
to one pair of protons is half that of the coupling to
another pair, so seven lines are seen instead of nine.
Sometimes a second derivative display can better
display slight changes in shape of the spectra and
improve resolution of hyperfine structure. Number 27
shows first- and second-derivative absorption and dis-
persion spectra of DPPH in fluid solution.
DUAL CAVITY AND g VALUES
In TIB 1962, Hyde described the dual cavity, which
basically is two TE102 cavities fastened together to
form a TE104 cavity, in which two samples can be
studied simultaneously with separate magnetic field
modulation coils. Because the two samples are at the
same microwave frequency, the dual cavity facilitates
comparison of g values, hyperfine splittings, and rel-
ative spin concentrations. The limitations of the dual
cavity are magnetic field differences at the locations
of the two samples and distortions of the microwave
field by quartz sample tubes, dewars, or differences in
solvents. Dual cavities were also discussed in TIB
1965 and in Randolph (26). Number 28 shows the
comparison of g values of Fremy’s salt and the tetra-
cene cation radical, using a dual cavity. The value
cited there for the g value of Fremy’s salt is that still
listed ((17); see p. 101) as a g-value standard. Using
a standard cavity on an E-3 spectrometer, g values
were measured accurately by sample replacement (no.
46) and accurate measurement of resonant frequencies
during each field scan. It was necessary to have the
spectrometer thermally stabilized to minimize drift to
get the most accurate results.
RADICALS GENERATED BY THERMAL
REACTIONS
Number 24 illustrated the power of EPR to identify,
via hyperfine splitting patterns, intermediates in
4 HYDE, EATON, AND EATON
chemical reactions. In this case the radical observed
was not the one that had been suggested in a proposed
reaction mechanism. Kinetics of oxidation of octade-
cene in the presence of an inhibitor were measured by
monitoring the EPR of a free radical intermediate
(no. 11).
PHOTOCHEMICALLY GENERATED
RADICALS
TIB 1959 showed the application of EPR to the study
of free radical intermediates produced photochemi-
cally, and introduced the potential user to three sig-
nificant advances in EPR instrumentation. Magnetic
field modulation at 100 kHz increased sensitivity by a
factor of 16 over previously used audiofrequency
modulation (200 Hz was used in TIB 1957). The
“multipurpose” cavity resonator had a grid that per-
mitted in situ ultraviolet irradiation of the sample.
Sample temperature was controlled by a nitrogen flow
system that has changed only slightly over the next
few decades (the article cited as “in press” is Piette
and Landgraf) (27). EPR is a major contributor to the
study of various steps in photosynthesis because many
of the species created during photosynthesis have
unpaired electrons (28 –30). Number 33 showed a
variety of signals generated by light in wild-type and
mutant algae.
TRIPLET RADICALS
When two unpaired electrons interact strongly
enough, singlet and triplet states can be produced. The
triplet state exhibits distinctive EPR spectra, which
were described in nos. 26 and 30, and applied in EPR
in the World of Biochemistry article. Number 26
presents the oriented single-crystal EPR spectrum of
the photoexcited triplet of naphthalene in durene,
which was used in the classic experiment by Hutchi-
son and Mangum (31, 32). The 1961 article presents
plots of the change in splitting upon rotation of the
crystal, but the actual spectra are shown in no. 26.
Later it was found that triplets can also be observed in
glassy solution. The ⌬ms ⫽ ⫾2 transition, at g ⫽ 4,
also called the half-field transition, was shown in no.
30. Photolysis of tryptophan, tyrosine, and phenylal-
anine in frozen aqueous solution yield triplet-state
EPR spectra for which the half-field transition and the
formation and decay rates are shown in EPR in the
World of Biochemistry.
Concepts in Magnetic Resonance Part A (Bridging Education and Research) DOI 10.1002/cmr.a
NITROXYL RADICAL SPIN PROBES
AND SPIN LABELS
Shortly after McConnell introduced the spin label
technique, nos. 39, 44, and 45 showed the community
how to use the effect of motion on the line shapes of
spin labels to monitor the uncoiling of a protein, the
binding of a nitrophenyl group to an antibody, and
changes in membrane structure and dynamics upon
other species interacting with the membrane. Spin
label and spin probe studies have become major
fields, encompassing fundamental studies of molecu-
lar motion (33), detailed structure of membranes (33–
37), distance measurements (38), fundamental studies
of electron spin relaxation in small molecules (39),
and many other biomolecular problems. Special atten-
tion should be paid to the power of site-directed spin
labeling, by which a spin label can be put at a selected
position in a protein (36, 40). Also, see multiple
chapters in the volumes of Biological Magnetic Res-
onance listed below, in the section on biological ap-
plications.
SEMIQUINONE RADICALS
Number 2 used the EPR spectra of semiquinone rad-
icals to illustrate the use of hyperfine splittings intro-
duced in no. 1. Number 4 showed the measurement of
decay of a semiquinone radical during repeated scans
through the spectrum. If the reaction time constants
were of the order of milliseconds, a stopped flow
system could be used, as shown in no. 18. Numbers
13 and 18 present a semiquinone spectrum obtained in
a chemical reaction. The article cited as “in press” in
no. 13 is Adams et al. (41). Many semiquinone radi-
cals have been studied (42). Recent advances in
stopped-flow EPR include the use of loop-gap and
dielectric resonators to decrease the impact of tran-
sients caused by stopping the fluid flow (43). Redox
of quinone species can result in a product (no. 3) with
an EPR spectrum similar to that of melanin-type rad-
icals. The EPR signals created in melanoma cells
upon irradiation or upon introducing air into the cell
suspension are shown in no. 42. Melanin EPR has
been studied extensively (44).
BIOLOGICAL APPLICATIONS
TIB 1961 cited the applications of EPR to biological
systems that had started with the 1954 studies by

Commoner et al. (45) and described applications to
cell metabolism (46) and enzyme-substrate reactions.
EPR of naturally occurring metal centers, of other
naturally occurring radicals, and of spin labels and
spin probes has made a major contribution to under-
standing structure and dynamics of biological sys-
tems. This development has been recorded in many
volumes in the book series Metal Ions in Biological
Systems (H. Sigel and A. Sigel, eds.) and Biological
Magnetic Resonance (L.J. Berliner et al., eds.). Re-
cent work on measuring distances between unpaired
electrons by EPR has also emphasized applications to
biological systems (38, 47). The scope of biological
applications of EPR is shown by volumes 23 and 24
of Biological Magnetic Resonance (48, 49). Two
spin-labeling volumes were edited by Berliner. Chap-
ters on EPR occur in volumes 1– 8 and 11 of Biolog-
ical Magnetic Resonance. Subsequent volumes have
tended to focus more on individual topics. The fol-
lowing volumes are about EPR:
13: EMR of Paramagnetic Molecules (1993)
14: Spin Labeling: The Next Millennium (1998 )
18: In Vivo EPR (ESR): Theory and Applications
(2003)
19: Distance Measurements in Biological Systems
by EPR (2000)
21: EPR: Instrumental Methods (2004)
22: Very High Frequency (VHF) ESR/EPR (2004 )
23: Biomedical EPR–Part A: Free Radicals, Met-
als, Medicine, and Physiology (2005)
24: Biomedical EPR–Part B: Methodology, Instru-
mentation, and Dynamics (2005)
Among the volumes of Metal Ions in Biological
Systems are:
22: ENDOR, EPR and Electron Spin Echo for
Probing Coordination Spheres (1987) (50)
30: Metalloenzymes Involving Amino Acid-Resi-
due and Related Radicals (1994) (51)
36: Interrelations Between Free Radicals and
Metal Ions in Life Processes (1999) (52)
ENZYMES AND ENZYME REACTIONS
Free radical intermediates in biochemical reactions
can be studied by EPR. Helmut Beinert provided the
example spectra reproduced in nos. 9 and 10. This
continues to be an active area of research (53–55).
The free radical reaction involved in the enzymatic
oxidation of chloropromazine by peroxidase was
shown to have a stoichiometry of two chloroproma-
zine and one H2O2. The article “in press” is Piette et
al. (56 ). Semiquinone and ascorbate radical interme-
Concepts in Magnetic Resonance Part A (Bridging Education and Research) DOI 10.1002/cmr.a
EPR AT WORK 5
diates were demonstrated in no. 19. See also
Yamazaki (57).
MANGANESE IN BIOLOGICAL SYSTEMS
The six-line Mn2⫹ EPR spectrum shown in no. 23
was also shown to exist in a culture of microorgan-
isms grown on a medium containing manganese,
showing uptake by the microorganisms without de-
structive elemental analysis (no. 6).
SENSITIVITY–AQUEOUS MN2ⴙ
Number 23 shows a spectrum of 10⫺6 M aqueous
Mn2⫹, which remains a challenging goal. Current
efforts to develop resonators and sample holders for
aqueous samples need to compare with this prior work
(11, 12, 58; see also the Bruker BioSpin AquaX
sample holder for aqueous samples). Note that the
aqueous Mn2⫹ spectrum was obtained with 20 G field
modulation, 180 mW incident on a TE102 cavity, and
1 s filter time constant.
BLOOD
It was observed that drying of blood resulted in for-
mation of methemoglobin, which exhibited EPR sig-
nals characteristic of iron (III), and also a sharp line
that was attributed to “breaking of the porphyrin ring
structure” (no. 8). Recent studies of various tissues are
described in Saifutdinov et al. (59).
Two of the articles titled “EPR in the World of
Biochemistry” also dealt with blood. The blood sera
of patients taking the drug phenothiazine yielded,
upon ultraviolet light irradiation, an EPR signal pro-
portional to the dose of the drug. The ascorbate rad-
ical is also produced during photolysis of blood se-
rum. It has a characteristic doublet splitting of 1.7 G.
OXIMETRY
In vivo measurement of oxygen is a goal that domi-
nates much of in vivo EPR instrumentation and meth-
odology development (60 – 63). Number 14, titled
“Disappearing Free Radicals,” foretold some of the
current efforts to develop carbonaceous materials
whose EPR signals are sensitive to oxygen (64, 65),
lithium phthalocyanine and related materials (66, 67),
and trityl radicals (68, 69).
6 HYDE, EATON, AND EATON
35 GHz
Thirty-five GHz was described in nos. 35, 36, and 38
and in TIB 1965/66. Thirty-five GHz was a practical
compromise between potential increased sensitivity
for samples of limited size, ease of sample handling,
quality of detectors, and so on. The advantages of
multifrequency EPR were described for understand-
ing poorly resolved spectra, cases in which the model
of the spectrum is tentative or uncertain, interpreting
relaxation times, and reducing second-order shifts. In
no. 35, it is shown that the g-anisotropy of DPPH is
large enough, relative to 14N hyperfine splittings, so
that spectra that appear symmetric at 9.5 GHz become
asymmetric at 35 GHz. It is also shown that the
molecular motion is fast enough to average the
anisotropies even at 35 GHz in toluene solution, but
not in heavy mineral oil, at room temperature. This
comparison, and the table of viscosity and frequency
dependence of g and hyperfine splittings, set out the
basis for future applications of multifrequency EPR to
the study of magnetic anisotropies and molecular mo-
tion.
The single-crystal spectra of Yb3⫹ in different sites
overlap at 9.1 GHz but are well-separated at 35 GHz,
making interpretation easier (no. 36). In no. 38, sin-
gle-crystal Mn2⫹ spectra from two nonequivalent
sites each yield five electronic transitions, and each of
these is split by the 55Mn nuclear spin (I ⫽ 5/2) into
six lines. At X-band the spectrum is further compli-
cated by forbidden transitions, but their relative inten-
sity is proportional to 1/B2, so the spectrum is easier
to interpret at 35 GHz than at 9.5 GHz.
Currently, although X-band (ca. 9 –10 GHz) still
dominates EPR practice, 35 GHz is considered a
normal frequency for CW EPR, and pulsed 35 GHz
EPR is now commercially available (Bruker Bio-
Spin). In addition, CW and pulsed 95 GHz EPR is
commercially available (Bruker BioSpin), and higher
frequencies are used in many labs (70 –75).
RARE EARTHS
Magnetic properties of rare earths relevant to EPR
were described in the TIB 1966 Spring and Fall is-
sues. Some g values were tabulated, and relaxation
properties were described. Rare earth EPR was dis-
cussed extensively by Abragam and Bleaney (76),
and in many reviews by Buckmaster (77, 78 ), Misra
(79, 80), and Low (81). The recent interest in Gd3⫹
complexes as MRI contrast agents has given promi-
nence to EPR studies of Gd3⫹ (82– 85).
Concepts in Magnetic Resonance Part A (Bridging Education and Research) DOI 10.1002/cmr.a
FERRO- AND ANTIFERROMAGNETISM
The study of strongly ferromagnetic or antiferromag-
netic materials by EPR, especially in concentrated
metal salts (as described in no. 34), metal clusters, or
alloys, has been a specialized subfield of EPR. Al-
though some studies can be performed on standard
EPR spectrometers, some investigators have found it
desirable to create spectrometers specifically for fer-
romagnetic resonance studies. See, for example, De-
nysenkov and Grishin (86). This is an active area of
research, in part because of the importance of mag-
netic thin films in information storage.
VANADYL ION
Numbers 5, 7, and 17 concern the spectra of the VO2⫹
(vanadyl) moiety. The observation of vanadium in
crude oil stimulated many other studies of fossil fuels
using EPR. Because the vanadium chemical environ-
ment in crude oil is substantially immobilized on the
EPR time scale, the spectrum displays a “powder
pattern” exhibiting the g anisotropy due to spin-orbit
coupling. In the vanadyl complex of acetyl acetonate
(VO(acac)2) in benzene solution at room temperature,
the g and hyperfine anisotropy is almost motionally
averaged. The remaining anisotropies are revealed in
the unequal line widths. This was described in detail
by Bruno et al. (87). Because of its long electron spin
relaxation time, the VO2⫹ ion is easy to study at room
temperature and was selected for early studies, but the
principles demonstrated by the study of VO2⫹ are
general. Biological aspects of vanadium EPR were
reviewed by Eaton and Eaton (88).
FORBIDDEN TRANSITIONS
Most EPR spectra observe transitions that are allowed
when the microwave B1 is perpendicular to the Zee-
man field. These transitions involve ⌬ms ⫽ ⫾1,
⌬mI ⫽ 0. Number 17 showed transitions in VO2⫹
spectra that have ⌬ms ⫽ ⫾1, ⌬mI ⫽ 1, normally
forbidden, which become partially allowed when B1
is parallel to the Zeeman field. They are more readily
seen for larger values of the ratio of the hyperfine to
the Zeeman field (A/g␤B0). The “in press” article is
Anderson and Piette (89). Study of forbidden transi-
tions has become an important area of metallobio-
chemistry because important oxidation states have
integer-spin states, such as S ⫽ 2 states of coupled
spin systems (90) and Fe2⫹ (91).

LIQUID CRYSTAL SOLVENTS
Because most paramagnetic species exhibit anisotro-
pic g and hyperfine interactions, an anisotropic sol-
vent, such as a liquid crystal, can at least partially
orient the metal or radical. As discussed in no. 40,
with known parameters, such as for the vanadyl ace-
tylacetonate complex, one can determine the degree
of alignment of the guest molecule and measure the
kinetics of phase transitions. In no. 41, the motion of
a dinitroxyl will not be isotropic in a liquid crystal,
and thus the dipolar interaction between spins will not
be averaged to zero as in an isotropic fluid, and the
lines split.
RELAXATION TIMES AND PASSAGE
EFFECTS
The title of no. 20, “High Sensitivity EPR as a Tool in
Solid State Research,” does not reveal that its topic is
saturation and passage effects, which are not re-
stricted to solid-state research. This example brought
to the attention of researchers some of the strange
effects that occur in the rapid passage regime (92) and
showed how to use these to estimate T1 without using
a pulsed EPR spectrometer. Pulsed saturation recov-
ery had been described previously (see references in
the chapter by Eaton and Eaton (93)), but a practical
spectrometer for measuring T1 in the range (a few
microseconds) described in no. 20 was not published
until 1974 –1975 (94, 95). Passage effects are com-
monly observed (see the article on EPR in Analytical
Instrumentation Handbook; (4)), and have been ex-
ploited from time to time. The rapid-passage disper-
sion display was essential for Feher’s ENDOR exper-
iments on donors in silicon (96), and this display
continues to be used in modern ENDOR studies (97,
98). This display was also used by Hyde and Dalton
(99) when they introduced the technique of saturation
transfer spectroscopy to measure very slow rotational
diffusion of spin-labeled molecules. More recent stud-
ies include the work of Harbridge et al. (100) and
Stoner et al. (101).
GAS-PHASE EPR
The gas-phase X-band spectrum of molecular oxygen
in no. 31 shows a vast number of transitions. These
sharp lines were obtained with 180 micron pressure of
O2. Oxygen in air at normal atmospheric pressure
exhibits much broader lines. A review of the gas-
Concepts in Magnetic Resonance Part A (Bridging Education and Research) DOI 10.1002/cmr.a
EPR AT WORK 7
phase spectra of oxygen and other small paramagnetic
molecules was published by Westenberg (102) and
Weltner (103, see chap. 4 and appendix D). For many
years certain O2 transitions have served as the S/N
standard for Q-band EPR. This caused some confu-
sion when a Q-band system was installed in Denver,
where the lower atmospheric pressure changed the
intensity and shape of the spectrum. As EPR spec-
trometers become more sensitive, one has to be aware
of the O2 spectrum even at X-band, and in some cases
it is necessary to purge the resonator with nitrogen to
avoid “background” problems due to normal room air.
RADICALS CREATED BY RADIATION
Radicals created by radiation are the subject of nos.
12, 15, 16, 25, and 47. It was pointed out that radicals
generated by ionizing energy irradiation could be
stabilized by using either low temperature (methyl
alcohol at 77 K was used in no. 15, and ice at 4 and
77 K was used in no. 16) or a solid. Radicals in
polymethylmethacrylate were shown in no. 12 and
L-glutamic acid was used in no. 15. From the hyper-
fine patterns, the structure of the radicals could be
proposed (no. 25). H atoms (ca. 526 G splitting) were
seen at 4 K but not at 77 K. In no. 47 it is shown that
the H atoms formed by radiation of ice migrated upon
thermal annealing at 195 K and reacted with thymine,
creating a radical whose identity could be inferred
from the hyperfine pattern. The article “in press” in
no. 47 is Holmes and Weiss (104 ). Enormous fields
of science have developed from this beginning
(105, 106 ).
EPR is used to determine whether foodstuffs have
been irradiated to preserve them. The radicals pro-
duced by irradiation of crystalline alanine are stable
enough to be used as an archival dosimeter, and a vast
number of articles have been published on the char-
acterization of the radicals in irradiated alanine (107).
See, for example, the special issues of Applied Radi-
ation and Isotopes in which articles presented at a
series of international conferences are published (108,
109). There is now an ASTM method for alanine
dosimetry.
Radiation damage in minerals, such as quartz, can
be used to date archeological artifacts and even geo-
thermal activity (107, 110).
Human exposure to radiation, especially acciden-
tal, can be measured via the EPR signals induced in
bones and teeth (106, 110 –113). The defect center
created in teeth by radiation is particularly stable.
Tooth dosimetry (human and animal), for example,
8 HYDE, EATON, AND EATON
was used to estimate the range of exposure caused by
the Chernobyl nuclear reactor accident.
SUMMARY PERSPECTIVE
The TIB and EPR at Work articles reproduced here
were written starting about a dozen years after the
discovery of EPR by Zavoisky (see Foundations of
Modern EPR (1)), by which time the Varian V4500
series spectrometers were available to researchers (the
first one shipped in 1956). The last article was written
in about 1968. The E-3 was introduced in 1966, and in
1967–1968 GRE used one to develop an undergrad-
uate laboratory at MIT based on electrochemical gen-
eration of radicals of substituted benzoquinones. The
E-line series, which implemented essentially all mod-
ern (but precomputer) CW EPR features, including a
circulator and reference arm in the bridge, was intro-
duced around 1970. By the early 1980s, Varian
stopped marketing EPR spectrometers. For the past
two decades, Bruker Instruments, now Bruker Bio-
Spin, has progressively introduced new EPR spec-
trometers for CW and pulsed EPR and ENDOR, not
only at X-band but also at higher (now to 95 GHz) and
lower (now to 1.1 GHz) frequencies. These new spec-
troscopic capabilities make possible new applications.
During the years covered by the EPR at Work
series, the foundations were laid for applications of
EPR in biochemistry, radiation chemistry, electro-
chemistry, solid-state physics, and in experimental
validation of the predictions of quantum mechanics of
odd-electron systems. Many applications of spin la-
beling, spin trapping (114, 115), in vivo EPR (60),
high-field/high-frequency EPR (75), electron spin
echo (116 –119) and FT EPR, and detailed studies of
electron spin relaxation times, all came since then.
The anisotropy of nitroxyl radical EPR, explored by
modern CW and pulsed EPR, has revealed much of
the current understanding of molecular motion in liq-
uids and glasses via many experiments in the Freed
lab (33). Calculation of EPR parameters is being
performed at a high level of sophistication (106, 120)
and has been incorporated into commercially avail-
able computational programs, such as Gaussian
(Wallingford, CT). The articles in Rudowicz et al.
(121) and Kawamori et al. (122) give an indication of
the scope of applications of modern EPR to physics
and magnetism, materials sciences, chemical reac-
tions, environmental sciences, biology and life sci-
ences, medical sciences, geology, and dosimetry, us-
ing CW, pulsed, and multiple-frequency EPR.
Many new technological improvements are on the
horizon (123). We predict that the future will see
Concepts in Magnetic Resonance Part A (Bridging Education and Research) DOI 10.1002/cmr.a
continued innovation (124) and hope that this review
of some of the fundamentals that stimulated EPR
applications in the early days will serve again to
stimulate new applications by students who come new
to the field.
ACKNOWLEDGMENTS
The preparation of this review was supported in part
by NIH grants NIBIB EB00557, EB002807, and P41
EB002034 (Prof. Howard Halpern, University of Chi-
cago, Chicago, IL) in Denver, and P41 EB001980 in
Milwaukee.
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BIOGRAPHIES
James S. Hyde received his B.S. and Ph.D.
in physics from MIT, the latter in 1959, and
accepted a position at Varian Associates in
the EPR group. He remained at Varian until
1975, and most of the material that is repro-
duced in the accompanying article was de-
veloped by him and his associates during that
16-year period. Since 1975, Dr. Hyde has
been professor of biophysics at the Medical
College of Wisconsin, where he serves as director of the National
Biomedical EPR Center. His awards include the Gold Medal of the
International Society of Magnetic Resonance in Medicine, the Gold
Medal of the International EPR/ESR Society, the Bruker Prize from
the EPR Discussion Group of the Royal Society of Chemistry
(London), and the Zavoisky Award, Kazan, Russia. He is a fellow
of the American Physical Society. He has written more than 350
articles and reviews, and he holds 32 U.S. Patents. He continues to
develop EPR technology, including digital detection strategies and
finite-element modeling of electromagnetic fields in novel EPR
resonators.
Sandra S. Eaton received her bachelor’s
degree at Wellesley College and obtained her
Ph.D. in inorganic chemistry at MIT in 1972.
She joined the faculty of the University of
Colorado at Denver in 1973. In 1990 she
moved to the Department of Chemistry and
Biochemistry at the University of Denver.
She teaches undergraduate and graduate-
level classes in physical and inorganic chem-
istry. In 1997, Gareth and Sandra jointly received the John Evans
Professorship at the University of Denver. In 2001 the EPR Dis-
cussion Group of the Royal Society of Chemistry (London)
awarded the Bruker Prize to them jointly. Their research program
involves continuous wave and pulsed EPR applied to the study of
relaxation times, spin-spin interaction, metal ions in biological
systems, and EPR imaging. They jointly organize the International
EPR Symposium, which is held in Colorado each summer. They
have jointly authored more 280 research articles, reviews, and book
chapters. They edited the books EPR Imaging and In Vivo EPR
(jointly with K. Ohno), Foundations of Modern EPR (jointly with
Kev M. Salikhov), Distance Measurements in Biological Systems
by EPR, Biomedical EPR–Part A: Free Radicals, Metals, Medicine,
and Physiology, and Biomedical EPR–Part B: Methodology, In-
strumentation, and Dynamics (jointly with Lawrence J. Berliner).
Gareth R. Eaton received his A.B. at Har-
vard and obtained his Ph.D. in inorganic
chemistry at MIT in 1972. Since then he has
been in the Department of Chemistry (now
the Department of Chemistry and Biochem-
istry) at the University of Denver. He was
promoted to professor in 1980. He served as
dean of natural sciences from 1984 to 1988
and as vice provost for research from 1988 to
1989. He teaches inorganic chemistry.
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Concepts in Magnetic Resonance Part A (Bridging Education and Research) DOI 10.1002/cmr.a