Magnetic Resonance Spectroscopy – EPR and NMR

A brief review of the relevant bits of quantum mechanics

1. Electrons have “spin”, - rotation of the charge about its axis generates a magnetic
field at each electron.
2. Electrons in orbitals with two electrons are spin-paired, - one with ms +½ (↑, α),
one with ms -½ (↓, β), - so that the spins and magnetic fields cancel (↑ ↓).
3. Most molecular bonds are formed by coalescence of atomic orbitals so as to
satisfy the lower energy state arising from spin-pairing. Most molecules therefore
have all orbitals occupied by magnetically “silent” electron pairs.
4. If an electron is added to or subtracted from such a molecule (by reduction or
oxidation) an unpaired electron is introduced which is not spin coupled, and
therefore acts as a magnet. Such molecules are said to be paramagnetic.
5. Some bonds have unpaired electrons, giving them an inherent paramagnetic
property.
6. In EPR spectroscopy, this property is measured to provide specific information
about the molecule and its environment.
7. Some nuclei also have magnetic properties, measured in NMR.
 Electron Paramagnetic Resonance (EPR) spectroscopy. What can we use it for?

• Assay of concentration and concentration changes: redox state of paramagnetic

species such as iron-sulfur clusters, semiquinones, flavin free-radicals,
cytochromes, P450, photosynthetic reaction center components, amino acid
radicals (Tyr, Trp), etc.
• Paramagnetic metals such as Mn2+, vanadyl ions (Mg2+-analogue) at catalytic
centers.
• Measurement of local polarity and local structural mobility using nitroxide spin
probes.
• Measurement of distance through spin-coupling with remote paramagnetic
centers.
• Measurement of structure through interaction with local nuclear spins. ENDOR
and pulsed methods (ESEEM) provide both distances and angles to give details
of structure in the reaction environment close to the paramagnetic center (NMR
through EPR).
• Measurement of electron distribution on paramagnetic species.
• Measurement of interactions (H-bonding) with local solvent (H2O).
Nuclear Magnetic Resonance (NMR) spectroscopy

1. The atomic nucleus is made up of protons (+-ve charge) and neutrons (neutral).
2. Like electrons, protons and neutrons (or nucleons) are quantum mechanical
entities, and their energetic properties can be described by operators, wave
functions, and quantum numbers.
3. Both protons and neutrons have a spin ½, with spin quantum number, mI,
which can have values of ↑ (up, +½, or α), or ↓ (down, -½, or β).
4. Total nuclear energy levels are lower if the constituent protons and neutrons
are spin-paired, so that for most nuclei, the spins (and magnetic fields) cancel.
Protons are spin-paired with protons, neutrons with neutrons.
5. Isotopes that have an odd number of either protons or neutrons have a net spin,
according to the Table below:
Number of protons Number of neutrons Nuclear spin, (I)
Even Even 0
Even Odd 1/2 or 3/2 or 5/2 …
Odd Even 1/2 or 3/2 or 5/2 …
Odd Odd 1 or 2 or 3 …
 What can we use NMR spectroscopy for?

Because of the wide range of nuclei that have spins, the applications of NMR
are more widespread than those of EPR. Resolution can be enhanced by
isotopic substitution for those nuclei that either are rare, or have no significant
population, at natural abundance.
• Identification and characterization of reaction products in chemistry through
characteristic chemical shifts in energy for particular nuclei.
• Proton exchange rates between -OH, -COOH, etc., and H2O
• Spin-spin coupling to identify bonding interactions.
• By extension of the above, - NMR can be used to obtain structures for
macromolecules at atomic resolution in solution.
• Magnetic Resonance Imaging (MRI), - a major advance in medical imaging of
internal tissues, - based on proton NMR.
 Spin and magnetism

Electrons and nuclei have magnetic properties only because they carry charges and
move. The magnetic force generated by a moving charge is proportional to the
current. In the case of a spinning entity, this translates into how fast it spins.
Why do different isotopes have different nuclear magnetic properties?
Since the nuclear charge is dependent on atomic number (the number of protons), and
different isotopes have the same atomic number (otherwise they are different
elements), they must all carry the same charge. Since there’s no change in charge on
adding neutrons, why is there a change in magnetic properties?
If a nucleus has an even number of protons and an even number of neutrons, all the
spins compensate, and the nucleus has no net spin. The nuclear magnetism arises
from the spin introduced by an extra neutron, which gives a net angular momentum.
The rate of spinning is dependent on angular momentum and therefore on mass. For
any particular mass, a given increment in spin on adding a neutron will start the whole
nucleus spinning at a rate dependent on the combined mass.
The magnetic difference between isotopes therefore depends in a complicated way on
the mass of the nucleus (the atomic weight), the number of charges it carries (the
atomic number), and how many unpaired nucleons are present.
 Some nuclear magnets of importance in biological studies

As we shall see later, the gyromagnetic ratio (γ) determines the energy at
which electromagnetic radiation will flip the spin of a nuclear magnet. A flip
occurs when the energy matches (or is in resonance with) the energy of the
transition. The energy needed (expressed in terms of frequency) depends on
the applied magnetic field (the strength of the magnet), - 4.7 Tesla in this
case.
 NMR ~10-200 mI = -½
MHz @ 4.7 T
EPR ~9.5GHz mI = ±½ γB0 h
@ 0.34 T Energy ∆E = hν =
(ν ) 2π
RT=0.002 kcal/mol
at 10oK
Energy of mI = +½
0
transition magnetic field strength (B0)

ν =E/h
(E = hν) ms = -½

RT=0.593 kcal/mol
at 298oK ms = ±½
Energy ∆E = hν = gβB0
ν - frequency Hz (ν )
ν~ - wavenumber cm-1
λ- wavelength nm ms = +½
0
1 ν
ν~ = = magnetic field strength (B0)
λ c
 On the nature of electromagnetic waves, and the transitions that
absorb or generate them

Our knowledge of electromagnetic waves comes from their interactions with

matter. We measure them by the transitions they generate in atoms, molecules, and
other antennae, and they are generated by excitation of transitions that decay by
emitting a photon. They are called electromagnetic, not because they are either
electrical or magnetic entities, but because they generate or are generated by
electrical transitions, which have a linked magnetic component.
The energy of photons is expressed in terms of frequency, wavenumber or
wavelength, all of which are related to energy through the Planck constant. All
photons move at the speed of light (~1ft/ns), - this relates frequency to wavelength.

The frequency is related to the timescale of the transition associated with

absorption or generation of the photon.
The transitions associated with EPR occur ν - frequency Hz
Energy of
with timescales in the ns range; those for ν - wavenumber cm-1
transition
NMR with timescales in the µs range.
λ- wavelength nm
These timescales are of importance in ν =E/h
terms of saturation, and the use of 1 ν
(E = hν) ν~ = =
pulsed techniques, as we will see later. λ c
 What is the difference between the electron and the nucleus as magnets?
1. The spin quantum numbers for the electron, proton and
neutron, all have the same value of ½. This is an intrinsic
angular momentum, - every electron, proton and neutron has
this property.
2. Because of the relation between angular momentum and mass,
a spin ½ body has a frequency of rotation determined by the
mass. Because nuclear particles (nucleons) have masses 1,836
× that of the electron, the nucleus spins much more slowly.
3. Because the magnetic field depends on the rate of rotation,
electrons have magnetic fields ~2,000 × that of protons, which
have higher fields than more massive nuclei. Hence magnetic
resonance occurs at much higher energies for EPR than NMR.
4. In addition to the angular momentum and magnetic properties arising from its
spin, an electron in a molecular orbital has an orbital angular momentum, which
also generates a magnetic field. The interaction between these two magnetic
fields is called spin-orbit coupling. The antiparallel arrangement (bottom, right)
has the lower energy, and is therefore favored. The electron magnetic field
depends on the total angular momentum, J. In general, both electrons and
nucleons spin-couple with other magnets in their environment.
Nuclear Magnetic Resonance mI = -½

In the absence of an external magnetic

mI = ±½ γB0 h
field, the spins of the nuclei are arranged Energy ∆E = hν =
(ν ) 2π
more or less at random. When a magnetic
field is applied, the spins align with the
field, just like bar magnets. However, mI = +½
because the energy involved is so small, 0
they can flip direction relatively easily. magnetic field strength (B0)

The consequence is that the energy level of the nucleus splits in a magnetic field,
as shown on the right. The population of spins in the mI +½ (α) state is (very
slightly) higher than in the mI -½ (β) state, because the energy is lower.
When photons of the “right” energy are absorbed, the spin of the
nucleus flips between the two states. If we look at the diagram, we can
see that the energy gap between the two states is dependent on the
strength of the applied magnetic field. As a consequence, photons are
absorbed which have a particular energy at a particular field. This is
what is meant by resonance.
γB0 h
∆E = hν =
2π
Here, γ is the gyromagnetic ratio for the nucleus, B0 is the magnetic
field strength, and h is Planck’s constant.
When we come to discuss pulsed NMR, we will need to refer to circular
motion. Here the preferred frame of reference is that of the circular
motion of precession. We define the precession or Larmor frequency:
ω = 2πυ
or by substitution in the above,
ω0 = γB0
 Some consequences of the energy scale.

The transitions leading to NMR absorption have energies in the radio frequency
range, depending on nucleus (γ) and the strength of the magnetic field generated by
the magnet.
NMR machines are rated by the frequency at which the proton is in NMR
resonance for the magnet they are built around, so we have 200 MHz, 500 MHz,
750 MHz and even 1 GHz NMR spectrometers. To achieve the higher fields, high
electrical currents are needed, which can be achieved using superconducting coils,
- these are generally called superconducting magnets (costing $M).
Since ω = 2πν = γB0, for a 200 MHz machine we need a magnet generating 4.7
Tesla; for a 500 MHz machine we need 11.74 Tesla, etc.
As we increase the energy gap (increase frequency), the small differences of
energy for transitions of nuclear magnets in different environments are also
increased, and our NMR spectrum will be better resolved. In addition, we also
increase the population difference for the two spin states as we increase the energy
(∆E/RT is increased), as discussed in the next slide.
These factors make a big difference in the amount of time needed to generate a
data set, - for example in solution of the structure of a protein.
The energy levels of the two spin states of importance in EPR and NMR are
relatively close. As we saw in previous slides, the ∆E values for the transitions are
much less than RT. As a consequence, the populations in the two states are almost
equal (see next slide for details). This means that we have to worry about the
phenomenon of stimulated emission.

Photons are promiscuous, - they will take advantage of any transition that matches
their energy. A “down” transition has the same energy gap as an “up” transition, so a
photon can be adsorbed, and promote a transition, in either direction. The laws of
energy conservation require that the energy absorbed must be given up, together
with the energy of the transition. As a consequence, two photons are emitted, of
equal energy to the one absorbed, to give one emitted net. If our two spins are in
almost equal populations, all the  spins will absorb one photon, and all the  spins
will emit one photon. We therefore do not see these changes in population.
As we have seen, the energy of electromagnetic waves is generally expressed in
frequency, but energy scales are all related through Planck’s constant and the speed
of light, so we can express these energies in J/mol, eV, or any other energy units.
For a particular temperature, any transition can be described in terms of an
equilibrium between two states. For example, when we flip the energy level of a
proton in a magnetic field, we have two states, ↑ or α, and ↓ or β, separated by ∆E,
calculated as above. Let us use Nα and Nβ to represent the relative populations in the
two states. We can represent the equilibration between these two states by Nα ' Nβ .
Then the ratio of these populations is given by the Boltzman distribution:
Nα / Nβ = e ∆E / RT
Remembering that the energies of the transitions associated with NMR (and those of
EPR) are both very much less than RT, we can see that only a very small excess of
states will be in the lower energy level. For the proton at 500 MHz, at ~25oC, since E
= hν, and Planck and Avagadro constants are 6.626.10-34 J.s and 6.02.1023 mol-1
respectively, energies in the RF range (~500 MHz) are 0.2 J/mol.
Nα / Nβ = 1.00008
It is these one in ten thousand spins that are available to absorb a photon to
provide an NMR signal.
 Electron Paramagnetic Resonance ms = -½

The same general considerations apply to EPR.

The photon energy for resonance depends on ms = ±½
Energy ∆E = hν = gβB0
the applied field. The field aligns the spins, to (ν )
give a splitting of the electron energy levels,
with a population in the ms +½ state that is
greater than that in the ms -½ state. Absorption ms = +½
0
of a photon at the resonance energy flips the magnetic field strength (B0)
spins. The net absorbance is due to transitions
from ms +½ to ms -½ levels.
Absorption of microwaves
changes the energy level so that
the small fraction of spins in the
lower energy state are flipped into
the opposite orientation.
Saturation occurs when flips in
both directions occur with equal
probability. The power needed to
saturate depends on the relaxation
time of the spin transition.
No field External field applied
The general principles of EPR and NMR are essentially the same, but
with the differences in energies already discussed.
However, the nomenclature is different, mainly for historical reasons.
The resonance condition for EPR is described by the equations:
E = gβB0ms
∆E = hν = E+ 1 − E− 1 = gβB0
2 2

Here, g, the dimensionless Landé g-value, describes the resonance

energy. Values around 2.003 are found for simple free-radical
systems. The term β is the Bohr magneton:
β = eh/(4π me) = 927.4 x 10-26 JT-1 in SI units, or
βν = β/h = e/(4π me) = 13.996 x 109 Hz T-1 in frequency units, or
βeV = β /(e/C) = 5.7884 x 10-5 eV T-1 in electron volt units, where (e/C)
is the electron charge, 1.602 x 10-19 C.
 Two different approaches to magnetic spectroscopy, CW and pulsed methods

Continuous Wave (CW)

The CW approach is that used in most EPR
spectrometers. It was the method used in
earlier NMR spectrometers, but has now
been supplanted by Fourier Transform
NMR (FT-NMR), which is a pulsed method
that we will discuss later. The new
generation of EPR spectrometers are also
pulse-based instruments.
In CW-EPR, the absorbance spectrum of the
sample is measured over a range of energies
appropriate for the magnetic field strength
of the magnet used. However, for technical
reasons, the spectrum is obtained by using a
constant microwave frequency, and varying
the magnetic field strength.
The spectrum is usually presented as the
first derivative of the absorption spectrum.
 Comparison of an EPR spectrometer, and a UV-VIS spectrophotometer

In this comparison, several points

are noteworthy.
1. In a spectrophotometer, the
spectrum is generated by
varying the wavelength. As we
just discussed, in a CW-EPR
spectrometer, we use a fixed
wavelength (frequency), but
vary the magnetic field. This is
achieved by the field scan and
modulation coils associated
with the magnet.

2. In the spectrophotometer shown, the light-beam is chopped by a rotating sector.

This gives it a period function, and allows use of phase and frequency sensitive
detection electronics, which improve the signal to noise ratio.
3. In the CW-EPR
spectrometer, the signal is
modulated at 100 kHz by
modulation of the
magnetic field. This
allows use of frequency
and phase sensitive
amplification to improve
the S/N ratio. The
intensity and modulation
frequency are monitored
to provide a reference
channel. The output signal
is an amplified difference.

Because of the use of a modulated signal and frequency selective amplification, the
detection electronics only respond to a change in absorbance in the sample. As a
consequence, the spectrum is output as the first derivative of the absorbance
spectrum.
Choice of EPR range
Use of a fixed microwave frequency and a varying field in CW-EPR is determined
by the difficulty of generating microwaves of sufficient intensity. The choice was
originally dependent on the development of radar technology. Radar uses
microwaves, with different frequencies for different purposes, and also needs a lot
of power. Although alternative technologies are now available, the design of EPR
spectrometers was based on the availability of klystron microwave sources from
the radar applications, with the frequencies below.

EPR spectroscopists talk about W-

band, Q-band, S-band, etc., EPR
spectrometers. Most work reported has
been done at X-band, with microwave
frequencies of ~9.5 GHz, and magnetic
field strength ~3400 Gauss, or 0.34
Tesla.
 EPR tubes
electronics
microwave bridge

magnet

computer for data sample

analysis