Analytical Biochemistry 529 (2017) 4e9

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Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio

Review article

Introduction to nuclear magnetic resonance

rik
Vladimír Mlyna
High Field MR Centre, Department of Biomedical Imaging and Image-Guided Therapy, Medical University of Vienna, 1090 Vienna, Austria

a r t i c l e i n f o a b s t r a c t

Article history: Nuclear magnetic resonance spectroscopy is a useful tool for studying normal and pathological
Received 10 March 2016 biochemical processes in tissues. In this review, the principles of nuclear magnetic resonance and
Received in revised form methods of obtaining nuclear magnetic resonance spectra are briefly outlined. The origin of the most
27 April 2016
important spectroscopic parametersdchemical shifts, coupling constants, longitudinal and transverse
Accepted 10 May 2016
Available online 19 May 2016
relaxation times, and spectroscopic line intensitiesdis explained, and the role of these parameters in
interpretation of spectra is addressed. Basic methodological concepts of localized spectroscopy and
spectroscopic imaging for the study of tissue metabolism in vivo are also described.
Keywords:
Nuclear magnetic resonance
© 2016 Elsevier Inc. All rights reserved.
Chemical shift
Coupling constant
Nuclear relaxation
Localized magnetic resonance spectroscopy

The theory of nuclear magnetic resonance (NMR or MR [mag-
netic resonance] in the biomedical field) and specific applications
in physics, chemistry, biology, and medicine are the subject of
numerous textbooks and monographs (e.g., Refs. [1e3]). This article
gives a short overview of the principles of NMR spectroscopy for
newcomers to the field and for those who have not used magnetic
resonance techniques as yet in their studies. It also provides pre-
requisites for the use of NMR spectroscopy in vivo for animal and
human studies.
Basic principles of nuclear magnetic resonance
The phenomenon of nuclear magnetic resonance was discov-
ered in 1946 by Purcell and Bloch. It is based on the interaction of
magnetic moments of nuclei of various atoms with magnetic fields.
The magnetic moment of nuclei is associated with a nuclear spin,
which is a form of angular momentum possessed by these nuclei.
The value of nuclear spin is defined by a spin number. The nuclear
magnetic moment associated with the nuclear spin depends on
properties of a nucleus and on its spin number. The nuclei of an
even number of protons and neutrons have zero nuclear spin and
magnetic moment, whereas those with an odd number of protons
Abbreviations used: NMR, nuclear magnetic resonance; MR, magnetic
resonance; RF, radiofrequency; FID, free induction decay.
E-mail address: vladimir.mlynarik@meduniwien.ac.at.
or neutrons possess a non-zero spin and magnetic moment. Among
those nuclei with an odd number of either protons or neutrons,
some nuclei, such as 1H, 31P, 13C, and 15N, have a spin number of ½,
which is favorable for practical applications of magnetic resonance.
If the spin state of these nuclei is measured individually in the
magnetic field, two spin states are observed that correspond to the
magnetic quantum numbers of ½ and e½. One of these states
corresponds to the orientation of nuclei parallel to the magnetic
field, and the other corresponds to its antiparallel orientation.
These two spin states have different energies. Note that, from the
point of view of quantum physics, it does not mean that the nuclei
in an object are in pure quantum states of ½ and e½.
In the static magnetic field, the nuclei with the non-zero
magnetic moment tend to assume the orientation with the
lowest energy. This orientation is, however, disturbed by thermal
energy. As a result, we can imagine that the orientation of the
nuclear moments in the magnetic field is almost random in space
with a slightly increased probability of their orientation in the
direction of the field, that is, with lower energy [4]. Such an
orientation of nuclear magnetic moments in the static magnetic
field of the induction B0 gives rise to macroscopic magnetization M
as a vector sum of individual nuclear magnetic moments. In
equilibrium, the magnetization M0 is aligned with the direction of
the static magnetic field.
The magnetization is a vector quantity, which obeys the rules of
classical electrodynamics [5]. Thus, the interaction of the magnetic

http://dx.doi.org/10.1016/j.ab.2016.05.006
0003-2697/© 2016 Elsevier Inc. All rights reserved.
 rik / Analytical Biochemistry 529 (2017) 4e9
V. Mlyna 5

fields with the magnetization can be described by the equation of

classical physics:

dM=dt ¼ gM  B ¼ M  u; (1)

where g is the gyromagnetic constant, which is defined as a ratio of

the nuclear magnetic moment to its angular momentum. According
to Eq. (1), if the magnetization is aligned with the magnetic field B0
(conventionally aligned with the z-axis), the vector product M  B0
is equal to zero and M is static (Fig. 1A). However, if M and B0 are
not parallel, the magnetization precesses about the direction of B0
with an angular frequency of u (u ¼ 2pn, where n is the frequency in
Hz), which is usually called the Larmor frequency (Fig. 1B). The
typical magnitude of B0 is several Tesla (T), and the Larmor fre-
quency usually lies in the 50e900-MHz range.
The magnetization M can be detected only when it is not
static, that is, when it precesses about the direction of the
magnetic field. According to Faraday's law, the moving
magnetization induces electromotive force in the coil, which is Fig.2. Definition of the rotating frame of coordinates x0 y0 z0 . The frame rotates around
the z-axis of the laboratory frame with the frequency u.
proportional to dM/dt. Thus, to measure the magnetization, we
need to tilt it away from being parallel with the magnetic field,
which will lead to its precession. Because a sudden tilting of the
static magnetic field is not technically feasible, the concept of orders of magnitude smaller than B0; thus, the typical frequency
resonance must be used. Indeed, a low-amplitude radiofrequency of the precession lies in the kHz range.
(RF) magnetic field B1 produced by an RF electric current in the By switching the B1 field on for several milliseconds, M is tilted
coil with the axis perpendicular to B0 is able to rotate the toward the x0 y0 plane (Fig. 4B). The application of the B1 field for a
magnetization. short time, t, is called the RF pulse. After turning B1 off, the tilted
Because the effect of a radiofrequency magnetic field on the magnetization precesses about B0 and induces a decaying electro-
precessing magnetization is hard to visualize in the laboratory motive force in the coil, which is called free induction decay (FID) or
frame, here we use a frame rotating around the axis parallel to NMR signal. The RF pulse can rotate the magnetization by an arbi-
the static magnetic field B0 with the Larmor frequency u (Fig. 2). trary angle (e.g., 45 , 90 , or 180 ). The RF pulse rotating the
In this frame of coordinates, the precession of the magnetization magnetization from the equilibrium state by  90 is called the
is fully compensated by rotation of the frame, which means that excitation pulse, and the 180 pulse is called the inversion pulse.
the magnetization is static in this frame at any angle between M If the RF pulse is short enough (on the order of milliseconds or
and B. In other words, the magnetization behaves as if there were shorter), it is capable of tilting not only the magnetization with
no static field in the rotating frame (Fig. 3). However, the RF field exactly the same Larmor frequency but also, in a similar way, the
with the Larmor frequency oriented perpendicular to B0 can be magnetization with a resonance frequency in the range of several
depicted as a static vector B1 in the rotating frame, for example, hundred or thousand Hz from that of the RF pulse. This property is
along the x0 -axis (Fig. 4A). The movement of the magnetization in useful for measuring MR spectra of complex molecules that contain
the rotating frame under the effect of the RF field B1 is also numerous peaks with different frequencies. When a uniform
described by Eq. (1), which means that it will precess about B1 excitation of a range of frequencies is required, amplitude-
parallel to the x0 -axis. The amplitude of the B1 field is several modulated (shaped) RF pulses can be used. The common pulse

Fig.1. Behavior of the magnetization M in the static magnetic field B0. M is static when parallel with B0 (A); otherwise, it precesses around the direction of the static magnetic
field (B).
6 rik / Analytical Biochemistry 529 (2017) 4e9
V. Mlyna

Fig.3. Visualization of magnetization M in the laboratory (A) and in the rotating frame of coordinates (B). The magnetization is static in the rotating frame, as if B0 were 0.

Fig.4. Visualization of the RF magnetic field in the rotating frame (A) and its effect on M, which is rotated toward the transverse plane (x0 y0 ). The angle of rotation depends on the
amplitude of the RF field, B1, and its duration t (B).

shapes and the corresponding excitation bandwidths are shown in an exponential course with the time constant called the longitudi-
Fig. 5. nal or spinelattice relaxation time T1. Due to a small energy differ-
ence between the spin quantum states ½ and e½, a spontaneous
Nuclear relaxation

The magnetization after an RF pulse is no longer the equilibrium

magnetization because its component parallel to the magnetic field
is reduced and there is a non-zero component of the magnetization
in the plane perpendicular to the direction of the static magnetic
field (the transverse plane). As mentioned above, the precessing
component in the transverse plane decays in time, which is caused
by the interaction of individual magnetic moments with local
magnetic fields on a molecular level. Such an interaction leads to a
loss of coherence (dephasing) of the components of transverse
magnetization. The decay of the magnetization in the transverse
plane is called transverse relaxation or spinespin relaxation (Fig. 6A).
This decay is generally exponential, and its time constant is called
the transverse relaxation time T2.
In practice, the transverse magnetization decays with the time
constant shorter than T2. This is due to an inhomogeneous static
magnetic field over the volume that provides the MR signal. The time
constant of the MR signal decay in a real magnet is denoted T2*.
After the application of an RF pulse, the component of the
magnetization parallel with B0 is reduced, fully removed or inver-
ted and begins to recover to its equilibrium state M0. This process is Fig.5. Various shapes of RF pulses and the corresponding frequency bandwidths that
called longitudinal or spinelattice relaxation (Fig. 6B). Again, there is they excite.
 rik / Analytical Biochemistry 529 (2017) 4e9
V. Mlyna 7

.
d ¼ n  nref nref (2)

The d values are usually expressed in ppm. Here, n is the reso-

nance frequency in Hz of a spectroscopic line of a measured com-
pound, and nref is the resonance frequency of a reference compound
in the same static magnetic field. Conventionally, the resonance
frequency of 1H nuclei of tetramethylsilane is used as a reference.

Indirect spinespin coupling

Another phenomenon that affects the appearance of NMR

Fig.6. Transverse (A) and longitudinal (B) relaxation of the magnetization. The
transverse magnetization decays to zero with the time constant T2, whereas the lon-
gitudinal magnetization recovers to the equilibrium value M0 with the time
constant T1.
transition from the higher to lower energy spin quantum state is
extremely rare. Thus, recovery of the equilibrium magnetization is
possible only through stimulated transitions, that is, through
interaction of nuclear magnetic moments with local fluctuating
magnetic fields. Consequently, only fluctuating magnetic fields
with a frequency close to the resonance frequency can cause lon-
gitudinal relaxation. Due to the fast reorientation of molecules in
liquids, the main sources of the microscopic fluctuating magnetic
fields are other neighboring nuclei with a non-zero magnetic
moment, predominantly other 1H nuclei in a molecule. This
relaxation mechanism is called dipolar relaxation.
Human or animal tissues are heterogeneous systems that
consist of a liquid phase with dissolved compounds of low molec-
ular weight and solid-like macromolecules. The backbones of these
macromolecules provide MR signals, which are not visible on
standard MR instruments. However, the macromolecules interact
with the liquid phase and affect the relaxation times of water and
dissolved metabolites. Thus, the typical T1 and T2 relaxation times
in body fluids are long and comparable to those in solutions
measured in high-resolution NMR spectroscopy. Conversely, T1 and
especially T2 relaxation times of tissues are much shorter than
those in solutions. The relaxation behavior substantially affects the
appearance of the spectra from biological tissues.
spectra is the indirect (scalar) spinespin coupling or J-coupling. This
effect is responsible for splitting spectroscopic lines into multi-
plets. The coupling of two nuclei or groups of nuclei is mediated
by the polarization of electrons on chemical bonds connecting
these nuclei. The form of polarization depends on the instant
orientation of the nuclear magnetic moments in the magnetic
field. The orientation of a nuclear moment parallel to the magnetic
field can increase the electron density at a neighboring nucleus,
and the antiparallel orientation can decrease it. This effect is
mutual, which means that it is the same for both coupled nuclei
and groups of nuclei. The magnitude of this interaction is called
the coupling constant J and is expressed in Hz. Depending on the
number of neighboring nuclei, different splitting patterns are
observed. It can be easily shown that if the difference in resonance
frequencies of the coupled nuclei is large enough, the multiplicity
of the splitting of spectroscopic lines of spine½ nuclei is equal to
n þ 1, where n is the number of neighboring nuclei. Thus, 1H
atoms in two interacting CH groups with different resonance
frequencies provide two doublets with the same splitting. Simi-
larly, the coupling of a methyl CH3 group and a methylene CH2
group results in splitting the CH2 resonance peak into a quartet
and that of the CH3 group into a triplet (Fig. 7). The multiplet
structure of spectroscopic lines and the coupling constants
are useful parameters for the structural analysis of organic

NMR spectra and spectroscopic parameters

Chemical shift

It was observed that nuclei of the same species (e.g., 1H in a

molecule) can have slightly different resonance frequencies. These
differences originate from the fact that the nuclei are surrounded
by electrons. In the static magnetic field, the moving electrons
produce local magnetic fields oriented opposite to the external
field, thereby reducing the actual magnetic field at the nuclei. The
shielding effect of electrons, which decreases resonance fre-
quencies of nuclei, varies with the chemical environment and is,
therefore, characteristic of specific structural fragments of organic
compounds (e.g., methyl, methylene, or methine 1H nuclei) and
their substituents (e.g., OH, NH2, NH, COOH, CONH). Because the Fig.7. The splitting pattern of NMR peaks given by the CH3CH2 group. Their multi-
shielding effect is proportional to B0, a field-independent param- plicity is equal to n þ 1, where n is the number of 1H atoms on the neighboring carbon.
eter, chemical shift, has been defined in the form of the d scale: J is the coupling constant.
8 rik / Analytical Biochemistry 529 (2017) 4e9
V. Mlyna

compounds. The coupling constants are independent of the static

magnetic field and, in general, decrease with the number of
chemical bonds between the interacting nuclei.

Spectroscopic line intensity

The most important spectroscopic parameter for applications

in vivo is the integral intensity of the spectroscopic line. Because the
line intensity is proportional to the molar concentration of the
given 1H nuclei that contribute to this line, MR spectroscopy can be
useful for calculating molar concentrations of compounds present
in the measured tissue. Depending on measurement parameters,
the resulting intensities of spectroscopic lines may be affected by
relaxation. To increase the signal-to-noise ratio, the spectrum is
usually obtained as a sum of FIDs acquired after multiple excita-
tions. When the repetition time between two excitations is too
short, the magnetization of the nuclei with longer T1 relaxation
times does not have enough time to recover. This results in a
reduction of the peak intensity. Typically in measurements in vivo,
there is a time delay between excitation and signal acquisition. In
such cases, the intensity of the acquired MR signal can be affected
by the T2 relaxation time. Both of these effects need to be consid- Fig.9. Principle of excitation of a slice. Using a magnetic field gradient along the z-axis,
the resonance frequency of the nuclei in the object increases linearly along the z-axis.
ered when NMR spectroscopy is used to determine the metabolic
The shaped pulse of a specific frequency excites only the 1H nuclei having their
profile of a normal or pathological tissue. resonance frequency within the bandwidth of the pulse.
The appearance of the spectra of tissues measured in vivo differs
from that of small molecules in solutions. Because of the rapid
decay of the MR signal with the time constant T2*, the width of the Localized MR spectroscopy and spectroscopic imaging
spectroscopic lines of tissues is much larger than in solutions.
Consequently, the resolution of the spectra obtained in vivo is much To measure the spectra of a solution in a sample tube, a single RF
lower and the peaks of many metabolites overlap. In addition, the pulse with subsequent signal acquisition is usually enough.
spectra of tissues contain broad signals provided by nuclei of the Obtaining an MR spectrum from a selected region in an organ of a
outer parts of macromolecules or those of smaller macromolecules living subject is more complicated. To select the MR signal arising
with higher mobility. Despite these limitations, the spectra ob- from a predefined region, magnetic field gradients are used. These
tained in vivo provide unique information about the concentrations are produced by electric currents through auxiliary coils wound
of tissue metabolites, which may differ from that obtained from around the magnet bore in such a way that the magnetic field in the
tissue extracts [6] (Fig. 8). bore changes linearly along any of the magnet axes. As a result, the

Fig.8. Comparison of an 1H spectrum in vivo of rat brain at B0 ¼ 14.1 T (upper trace) with a spectrum in vitro of brain extract at B0 ¼ 11.7 T (lower trace). Note a decrease in the peak
of phosphocreatine (short arrow) and an increase in the lactate peak (long arrow) in the spectrum of the extract. (Spectrum in vitro reprinted with permission from Ref. [6]).
 rik / Analytical Biochemistry 529 (2017) 4e9
V. Mlyna
Fig.10. Intersection of three slice-selective pulses along different axes defines a
volume of interest, from which a localized spectrum is obtained.
resonance frequency of the nuclei along the given axis changes
linearly as well. If a shaped RF pulse, which is able to excite a certain
range of frequencies (see Fig. 5), is applied in the presence of the
field gradient, the magnetization will be excited only in a slice of an
object and will remain in the equilibrium state above and below
this slice (Fig. 9). The use of a shaped RF pulse together with a
magnetic field gradient is called a slice-selective pulse or slice-se-
lective excitation. The application of three orthogonal slice-selective
pulses excites the magnetization in three orthogonal slices that
intersect in a volume of interest (Fig. 10). The MR signal that has
been excited by the slice-selective pulses outside the volume of
interest is canceled or destroyed either by changing the phase of
the RF pulses or by applying additional field gradients for a short
time. As a result, the MR signal only from the volume of interest is
detected. Using this concept, a spectrum from only one selected
volume is usually measured at a time.
Another concept called phase encoding, which is commonly
used in MR imaging, can also be used for localization in MR
spectroscopy. It is based on switching the field gradient for a
short time, which produces a phase shift of the magnetization
component as a function of the distance of the nuclei from the
magnet center (Fig. 11). If the measurement of the spectrum is
repeated many times while increasing the magnetic field gradient
in small steps, the series of phase shifts of the transverse
magnetization produced by the nuclei at a specific distance de-
fines a specific frequency of rotation of the magnetization in the
9
Fig.11. Principle of phase encoding for spectroscopic imaging. The phase of the
transverse magnetization is changed during the application of the phase encoding
gradient Gphas. This phase change depends on the distance of the nuclei from the
magnet center.
transverse plane. Using a Fourier transform across the series of
spectra, spatial information about the position of the resonating
nuclei is obtained. This technique, called spectroscopic imaging,
provides a series of spectra of the measured organ in one, two, or
three spatial dimensions. Metabolite concentrations obtained
from the two- and three-dimensional data enable the construc-
tion of metabolic maps.
The excited magnetization can be further manipulated by
additional RF pulses, and its phase can evolve during short time
delays. By applying such pulse sequences, one can extract infor-
mation about individual metabolites (so-called spectroscopic
editing) or suppress unwanted peaks in the spectra.
References
[1] J. Keeler, Understanding NMR Spectroscopy, second ed., John Wiley, Chichester,
UK, 2010.
[2] R.A. de Graaf, In Vivo MR Spectroscopy: Principles and Techniques, second ed.,
John Wiley, Chichester, UK, 2007.
[3] J.D. Roberts, Nuclear Magnetic Resonance: Applications to Organic Chemistry,
McGraweHill, New York, 2006.
[4] L.G. Hanson, Is quantum mechanics necessary for understanding magnetic
resonance? Concepts Magn. Reson. A 32 (2008) 329e340.
[5] D.I. Hoult, The magnetic resonance myth of radio waves, Concepts Magn. Reson.
1 (1989) 1e5.
[6] R.A. de Graaf, G.M. Chowdhury, K.L. Behar, Quantification of high-resolution 1H
NMR spectra from rat brain extracts, Anal. Chem. 83 (2011) 216e224.