peak is broadened owing to interactions of the unpaired electron with the rest of
the molecule (spin–lattice interactions). This allows further conclusions as to the
molecular structure.
High-resolution EPR may be performed by examining thehyperfine splittingof the
absorption peak which is caused by interaction of the unpaired electron with adjacent
nuclei, thus yielding information about the spatial location of atoms in the molecule.
The proton hyperfine splitting for free radicals occurs in the range of 0–3*10^3 T, and
yields data analogous to those obtained in high-resolution NMR (see Section 13.5).
The effective resolution of an EPR spectrum can be considerably improved by
combining the method with NMR, a technique calledelectron nuclear double resonance
(ENDOR). Here, the sample is irradiated simultaneously with microwaves for EPR and
radio frequencies (RF) for NMR. The RF signal is swept for fixed points in the EPR
spectrum, yielding the EPR signal height versus nuclear RF. This approach is particu-
larly useful when there are a large number of nuclear levels that broaden the normal
electron resonance lines.
The technique ofelectron double resonance(ELDOR) finds an application in the
separation of overlapping multiradical spectra and the study of relaxation phenomena,
for example chemical spin exchange. In ELDOR, the sample is irradiated with two
microwave frequencies simultaneously. One is used for observation of the EPR signal
at a fixed point in the spectrum, the other is used to sweep other parts of the spectrum.
The recorded spectrum is plotted as a function of the EPR signal as a function of the
difference of the two microwave frequencies.13.4.4 Instrumentation
Figure 13.5 shows the main components of an EPR instrument. The magnetic fields
generated by the electromagnets are of the order of 50 to 500 mT, and variations of
less than 10^6 are required for highest accuracy. The monochromatic microwave
radiation is produced in aklystronoscillator with wavelengths around 3 cm (9 GHz).
The samples are required to be in the solid state; hence biological samples are
usually frozen in liquid nitrogen. The technique is also ideal for investigation of
membranes and membrane proteins. Instead of plotting the absorption A versusB,
it is the first-order differential (dA/dB) that is usually plotted againstB(Fig. 13.6).
Such a shape is called a ‘line’ in EPR spectroscopy. Generally, there are relatively
few unpaired electrons in a molecule, resulting in fewer than 10 lines, which are not
closely spaced.13.4.5 Applications
Metalloproteins
EPR spectroscopy is one of the main methods to study metalloproteins, particularly
those containing molybdenum (xanthine oxidase), copper (cytochrome oxidase, copper
blue enzymes) and iron (cytochrome, ferredoxin). Both copper and non-haem iron,
which do not absorb in the UV/Vis region, possess EPR absorption peaks in one532 Spectroscopic techniques: II Structure and interactions