What is NMR?

Nuclear magnetic resonance spectroscopy, most commonly

known as NMR spectroscopy or magnetic resonance
spectroscopy (MRS), is a spectroscopic technique to observe
local magnetic fields around atomic nuclei.
It is a spectroscopy technique that is based on the absorption of
electromagnetic radiation in the radiofrequency region 4 to 900 MHz
by nuclei of the atoms.
Over the past fifty years, NMR has become the preeminent
technique for determining the structure of organic compounds.
Of all the spectroscopic methods, it is the only one for which a
complete analysis and interpretation of the entire spectrum is
normally expected.

Principle of Nuclear Magnetic Resonance

(NMR) Spectroscopy
1. The principle behind NMR is that many nuclei have spin and all
nuclei are electrically charged. If an external magnetic field is
applied, an energy transfer is possible between the base energy to
a higher energy level (generally a single energy gap).
2. The energy transfer takes place at a wavelength that corresponds
to radio frequencies and when the spin returns to its base level,
energy is emitted at the same frequency.
3. The signal that matches this transfer is measured in many ways
and processed in order to yield an NMR spectrum for the nucleus
concerned.
 Working of Nuclear Magnetic Resonance
(NMR) Spectroscopy
 The sample is placed in a magnetic field and the NMR signal is
produced by excitation of the nuclei sample with radio
waves into nuclear magnetic resonance, which is detected with
sensitive radio receivers.
 The intramolecular magnetic field around an atom in a molecule
changes the resonance frequency, thus giving access to details of
the electronic structure of a molecule and its individual functional
groups.
 As the fields are unique or highly characteristic to individual
compounds, NMR spectroscopy is the definitive method to identify
monomolecular organic compounds.
 Besides identification, NMR spectroscopy provides detailed
information about the structure, dynamics, reaction state, and
chemical environment of molecules.
 The most common types of NMR are proton and carbon-13
NMR spectroscopy, but it is applicable to any kind of sample that
contains nuclei possessing spin.
Instrumentation of Nuclear Magnetic
Resonance (NMR) Spectroscopy
1. Sample holder
Glass tube with 8.5 cm long, 0.3 cm in diameter.
2. Permanent magnet
It provides a homogeneous magnetic field at 60-100 MHZ
3. Magnetic coils
These coils induce a magnetic field when current flows through
them
4. Sweep generator
To produce an equal amount of magnetic field pass through the
sample
5. Radio frequency transmitter
A radio transmitter coil transmitter that produces a short powerful
pulse of radio waves
6. Radio frequency receiver
A radio receiver coil that detects radio frequencies emitted as nuclei
relax to a lower energy level
7. Read out systems
A computer that analyses and records the data.
 Applications of Nuclear Magnetic
Resonance (NMR) Spectroscopy
Spectroscopy is the study of the interaction of electromagnetic
radiation with matter. NMR spectroscopy is the use of the NMR
phenomenon to study the physical, chemical, and biological
properties of matter.
 It is an analytical chemistry technique used in quality control.
 It is used in research for determining the content and purity of a
sample as well as its molecular structure. For example, NMR can
quantitatively analyze mixtures containing known compounds.
 NMR spectroscopy is routinely used by chemists to study chemical
structure using simple one-dimensional techniques. Two-
dimensional techniques are used to determine the structure of
more complicated molecules.
 These techniques are replacing x-ray crystallography for the
determination of protein structure.
 Time domain NMR spectroscopy techniques are used to probe
molecular dynamics in solution.
 Solid state NMR spectroscopy is used to determine the molecular
structure of solids.
 Other scientists have developed NMR methods-of measuring
diffusion coefficients.

Basic NMR techniques

Resonant frequency
When placed in a magnetic field, NMR active nuclei (such as 1H or 13C)
absorb electromagnetic radiation at a frequency characteristic of the isotope.[8] The resonant
frequency, energy of the radiation absorbed, and the intensity of the signal are proportional to the
strength of the magnetic field. For example, in a 21 Tesla magnetic field, hydrogen nuclei (commonly
referred to as protons) resonate at 900 MHz. It is common to refer to a 21 T magnet as a
900 MHz magnet since hydrogen is the most common nucleus detected, however different nuclei will
resonate at different frequencies at this field strength in proportion to their nuclear magnetic
moments.

Sample handling
An NMR spectrometer typically consists of a spinning sample-holder inside a very strong
magnet, a radio-frequency emitter, and a receiver with a probe (an antenna assembly) that goes
inside the magnet to surround the sample, optionally gradient coils for diffusion measurements, and
electronics to control the system. Spinning the sample is usually necessary to average out
diffusional motion, however some experiments call for a stationary sample when solution movement
is an important variable. For instance, measurements of diffusion constants (diffusion ordered
spectroscopy or DOSY)[9][10] are done using a stationary sample with spinning off, and flow cells can
be used for online analysis of process flows.

Deuterated solvents
The vast majority of molecules in a solution are solvent molecules, and most regular solvents
are hydrocarbons and so contain NMR-active hydrogen-1 nuclei. In order to avoid having the signals
from solvent hydrogen atoms overwhelm the experiment and interfere in analysis of the dissolved
analyte, deuterated solvents are used where 99+% of the protons are replaced
with deuterium (hydrogen-2).[11] The most widely used deuterated solvent
is deuterochloroform (CDCl3), although other solvents may be used for various reasons, such as
solubility of a sample, desire to control hydrogen bonding, or melting or boiling points. The chemical
shifts of a molecule will change slightly between solvents, and therefore the solvent used will almost
always be reported with chemical shifts.[citation needed] Proton NMR spectra are often calibrated against the
known solvent residual proton peak as a secondary standard instead of adding tetramethylsilane
(defined as a chemical shift of zero).

Shim and lock

To detect the very small frequency shifts due to nuclear magnetic resonance, the applied
magnetic field must be constant throughout the sample volume. High resolution NMR spectrometers
use shims to adjust the homogeneity of the magnetic field to parts per billion (ppb) in a volume of a
few cubic centimeters. In order to detect and compensate for inhomogeneity and drift in the
magnetic field, the spectrometer maintains a "lock" on the solvent deuterium frequency with a
separate lock unit, which is essentially an additional transmitter and RF processor tuned to the lock
nucleus (deuterium) rather than the nuclei of the sample of interest. [12] In modern NMR spectrometers
shimming is adjusted automatically, though in some cases the operator has to optimize the shim
parameters manually to obtain the best possible resolution

Acquisition of spectra
Upon excitation of the sample with a radio frequency (60–1000 MHz) pulse, a nuclear
magnetic resonance response - a free induction decay (FID) - is obtained. It is a very weak signal,
and requires sensitive radio receivers to pick up. A Fourier transform is carried out to extract the
frequency-domain spectrum from the raw time-domain FID. A spectrum from a single FID has a
low signal-to-noise ratio, but it improves readily with averaging of repeated acquisitions. Good 1H
NMR spectra can be acquired with 16 repeats, which takes only minutes. However, for elements
heavier than hydrogen, the relaxation time is rather long, e.g. around 8 seconds for 13C. Thus,
acquisition of quantitative heavy-element spectra can be time-consuming, taking tens of minutes to
hours.
 Following the pulse, the nuclei are, on average, excited to a certain angle vs. the
spectrometer magnetic field. The extent of excitation can be controlled with the pulse width, typically
ca. 3-8 µs for the optimal 90° pulse. The pulse width can be determined by plotting the (signed)
intensity as a function of pulse width. It follows a sine curve, and accordingly, changes sign at pulse
widths corresponding to 180° and 360° pulses.
Decay times of the excitation, typically measured in seconds, depend on the effectiveness of
relaxation, which is faster for lighter nuclei and in solids, and slower for heavier nuclei and in
solutions, and they can be very long in gases. If the second excitation pulse is sent prematurely
before the relaxation is complete, the average magnetization vector has not decayed to ground
state, which affects the strength of the signal in an unpredictable manner. In practice, the peak areas
are then not proportional to the stoichiometry; only the presence, but not the amount of functional
groups is possible to discern. An inversion recovery experiment can be done to determine the
relaxation time and thus the required delay between pulses. A 180° pulse, an adjustable delay, and
a 90° pulse is transmitted. When the 90° pulse exactly cancels out the signal, the delay corresponds
to the time needed for 90° of relaxation.[15] Inversion recovery is worthwhile for quantitative 13C, 2D
and other time-consuming experiments.

Chemical shift
A spinning charge generates a magnetic field that results in a magnetic moment proportional
to the spin. In the presence of an external magnetic field, two spin states exist (for a spin 1/2
nucleus): one spin up and one spin down, where one aligns with the magnetic field and the other
opposes it. The difference in energy (ΔE) between the two spin states increases as the strength of
the field increases, but this difference is usually very small, leading to the requirement for strong
NMR magnets (1-20 T for modern NMR instruments). Irradiation of the sample with energy
corresponding to the exact spin state separation of a specific set of nuclei will cause excitation of
those set of nuclei in the lower energy state to the higher energy state.
For spin 1/2 nuclei, the energy difference between the two spin states at a given magnetic
field strength is proportional to their magnetic moment. However, even if all protons have the same
magnetic moments, they do not give resonant signals at the same frequency values. This difference
arises from the differing electronic environments of the nucleus of interest. Upon application of an
external magnetic field, these electrons move in response to the field and generate local magnetic
fields that oppose the much stronger applied field. This local field thus "shields" the proton from the
applied magnetic field, which must therefore be increased in order to achieve resonance (absorption
of rf energy). Such increments are very small, usually in parts per million (ppm). For instance, the
proton peak from an aldehyde is shifted ca. 10 ppm compared to a hydrocarbon peak, since as
an electron-withdrawing group, the carbonyl deshields the proton by reducing the local electron
density. The difference between 2.3487 T and 2.3488 T is therefore about 42 ppm. However a
frequency scale is commonly used to designate the NMR signals, even though the spectrometer
may operate by sweeping the magnetic field, and thus the 42 ppm is 4200 Hz for a 100 MHz
reference frequency (rf).
However, given that the location of different NMR signals is dependent on the external
magnetic field strength and the reference frequency, the signals are usually reported relative to a
reference signal, usually that of TMS (tetramethylsilane). Additionally, since the distribution of NMR
signals is field dependent, these frequencies are divided by the spectrometer frequency. However,
since we are dividing Hz by MHz, the resulting number would be too small, and thus it is multiplied
by a million. This operation therefore gives a locator number called the "chemical shift" with units of
parts per million.[16] In general, chemical shifts for protons are highly predictable since the shifts are
primarily determined by simpler shielding effects (electron density), but the chemical shifts for many
heavier nuclei are more strongly influenced by other factors including excited states ("paramagnetic"
contribution to shielding tensor).
he chemical shift provides information about the structure of the molecule. The conversion of
the raw data to this information is called assigning the spectrum. For example, for the 1H-NMR
spectrum for ethanol (CH3CH2OH), one would expect signals at each of three specific chemical
shifts: one for the CH3 group, one for the CH2 group and one for the OH group. A typical CH3 group
has a shift around 1 ppm, a CH2 attached to an OH has a shift of around 4 ppm and an OH has a
shift anywhere from 2–6 ppm depending on the solvent used and the amount of hydrogen bonding.
While the O atom does draw electron density away from the attached H through their mutual sigma
bond, the electron lone pairs on the O bathe the H in their shielding effect.

In paramagnetic NMR spectroscopy, measurements are conducted on paramagnetic samples. The

paramagnetism gives rise to very diverse chemical shifts. In 1H NMR spectroscopy, the chemical
shift range can span up to thousands of ppm.

Because of molecular motion at room temperature, the three methyl protons average out during the
NMR experiment (which typically requires a few ms). These protons become degenerate and form a
peak at the same chemical shift.

The shape and area of peaks are indicators of chemical structure too. In the example above—the
proton spectrum of ethanol—the CH3 peak has three times the area of the OH peak. Similarly the
CH2 peak would be twice the area of the OH peak but only 2/3 the area of the CH3 peak.

Software allows analysis of signal intensity of peaks, which under conditions of optimal relaxation,
correlate with the number of protons of that type. Analysis of signal intensity is done by integration—
the mathematical process that calculates the area under a curve. The analyst must integrate the
peak and not measure its height because the peaks also have width—and thus its size is dependent
on its area not its height. However, it should be mentioned that the number of protons, or any other
observed nucleus, is only proportional to the intensity, or the integral, of the NMR signal in the very
simplest one-dimensional NMR experiments. In more elaborate experiments, for instance,
experiments typically used to obtain carbon-13 NMR spectra, the integral of the signals depends on
the relaxation rate of the nucleus, and its scalar and dipolar coupling constants. Very often these
factors are poorly known - therefore, the integral of the NMR signal is very difficult to interpret in
more complicated NMR experiments.

FIG. Example of the chemical shift: NMR spectrum of hexaborane B6H10 showing peaks shifted
in frequency, which give clues as to the molecular structure

{NMR Interpretation of hexaborane

12.8 MHz. range (right)
A simple example of the chemical shift is the "a" and "b" doublets (pairs of peaks). "A" and "b" are two
groups of boron atoms in different electronic environments, which place the "a" doublet to the right of the
"b" doublet in the spectrum.
The relative peak heights of the "a" and "b" doublets suggest a 1:5 ratio of borons in one environment to
borons in another environment.
This suggests, combined with the molecular weight of the molecule:

 5 boron atoms in one environment ("b") and

 1 boron atom in another environment ("a"),
which in turn suggests a pyramidal structure, which does appear in the atomic diagram.
40 MHz. range (left)
"a" suggests a hydrogen at the apex, which is in the atomic diagram sticking straight up.
"b" suggests hydrogens bonded to one boron each, which are in the atomic diagram sticking straight out
from the edges.
"c" suggests other bridge hydrogens, which is to say hydrogens in the middle of a boron-hydrogen-boron
bonding arrangement like a hydrogen bridge between two boron shores, which are in a ring around the
atomic diagram.}
J-coupling
Some of the most useful information for structure determination in a one-dimensional NMR spectrum
comes from J-coupling or scalar coupling (a special case of spin–spin coupling) between NMR
active nuclei. This coupling arises from the interaction of different spin states through the chemical
bonds of a molecule and results in the splitting of NMR signals. For a proton, the local magnetic field
is slightly different depending on whether an adjacent nucleus points towards or against the
spectrometer magnetic field, which gives rise to two signals per proton instead of one. These
splitting patterns can be complex or simple and, likewise, can be straightforwardly interpretable or
deceptive. This coupling provides detailed insight into the connectivity of atoms in a molecule.

Coupling to n equivalent (spin ½) nuclei splits the signal into a n+1 multiplet with intensity ratios
following Pascal's triangle as described on the right. Coupling to additional spins will lead to further
splittings of each component of the multiplet e.g. coupling to two different spin ½ nuclei with
significantly different coupling constants will lead to a doublet of doublets (abbreviation: dd). Note
that coupling between nuclei that are chemically equivalent (that is, have the same chemical shift)
has no effect on the NMR spectra and couplings between nuclei that are distant (usually more than 3
bonds apart for protons in flexible molecules) are usually too small to cause observable
splittings. Long-range couplings over more than three bonds can often be observed
in cyclic and aromatic compounds, leading to more complex splitting patterns.

For example, in the proton spectrum for ethanol described above, the CH3 group is split into
a triplet with an intensity ratio of 1:2:1 by the two neighboring CH2 protons. Similarly, the CH2 is split
into a quartet with an intensity ratio of 1:3:3:1 by the three neighboring CH3 protons. In principle, the
two CH2 protons would also be split again into a doublet to form a doublet of quartets by the hydroxyl
proton, but intermolecular exchange of the acidic hydroxyl proton often results in a loss of coupling
information.

Coupling to any spin-1/2 nuclei such as phosphorus-31 or fluorine-19 works in this fashion (although
the magnitudes of the coupling constants may be very different). But the splitting patterns differ from
those described above for nuclei with spin greater than ½ because the spin quantum number has
more than two possible values. For instance, coupling to deuterium (a spin 1 nucleus) splits the
signal into a 1:1:1 triplet because the spin 1 has three spin states. Similarly, a spin 3/2 nucleus such
as 35Cl splits a signal into a 1:1:1:1 quartet and so on.

Coupling combined with the chemical shift (and the integration for protons) tells us not only about the
chemical environment of the nuclei, but also the number of neighboring NMR active nuclei within the
molecule. In more complex spectra with multiple peaks at similar chemical shifts or in spectra of
nuclei other than hydrogen, coupling is often the only way to distinguish different nuclei.
 Multiplicit
Intensity ratio
y

Singlet (s) 1

Doublet (d) 1:1

Triplet (t) 1:2:1

Quartet (q) 1:3:3:1

Quintet 1:4:6:4:1

Sextet 1:5:10:10:5:1
Example H NMR spectrum (1-dimensional) of ethanol plotted
1
Septet 1:6:15:20:15:6:1
as signal intensity vs. chemical shift. There are three different
types of H atoms in ethanol regarding NMR. The hydrogen (H) on the −OH group is not
coupling with the other H atoms and appears as a singlet, but the CH − and the −CH − hydrogens
3 2

are coupling with each other, resulting in a triplet and quartet respectively.
Second-order (or strong) coupling

The above description assumes that the coupling constant is small in comparison with the difference
in NMR frequencies between the inequivalent spins. If the shift separation decreases (or the
coupling strength increases), the multiplet intensity patterns are first distorted, and then become
more complex and less easily analyzed (especially if more than two spins are involved).
Intensification of some peaks in a multiplet is achieved at the expense of the remainder, which
sometimes almost disappear in the background noise, although the integrated area under the peaks
remains constant. In most high-field NMR, however, the distortions are usually modest and the
characteristic distortions (roofing) can in fact help to identify related peaks.

Some of these patterns can be analyzed with the method published by John Pople,[18] though it has
limited scope.
 1

H NMR spectrum of menthol with chemical shift in ppm on the horizontal axis. Each magnetically
inequivalent proton has a characteristic shift, and couplings to other protons appear as splitting of the
peaks into multiplets: e.g. peak a, because of the three magnetically equivalent protons in methyl
group a, couple to one adjacent proton (e) and thus appears as a doublet.

Second-order effects decrease as the frequency difference between multiplets increases, so that
high-field (i.e. high-frequency) NMR spectra display less distortion than lower frequency spectra.
Early spectra at 60 MHz were more prone to distortion than spectra from later machines typically
operating at frequencies at 200 MHz or above.

Furthermore, as in the figure to the right, J-coupling can be used to identify ortho-meta-para
substitution of a ring. Ortho coupling is the strongest at 15 Hz, Meta follows with an average of 2 Hz,
and finally para coupling is usually insignificant for studies.

Magnetic in equivalence
More subtle effects can occur if chemically equivalent spins (i.e., nuclei related by symmetry and so
having the same NMR frequency) have different coupling relationships to external spins. Spins that
are chemically equivalent but are not indistinguishable (based on their coupling relationships) are
termed magnetically inequivalent. For example, the 4 H sites of 1,2-dichlorobenzene divide into two
chemically equivalent pairs by symmetry, but an individual member of one of the pairs has different
couplings to the spins making up the other pair. Magnetic inequivalence can lead to highly complex
spectra which can only be analyzed by computational modeling. Such effects are more common in
NMR spectra of aromatic and other non-flexible systems, while conformational averaging about C−C
bonds in flexible molecules tends to equalize the couplings between protons on adjacent carbons,
reducing problems with magnetic inequivalence.
Correlation spectroscopy
Correlation spectroscopy is one of several types of two-dimensional nuclear magnetic resonance
(NMR) spectroscopy or 2D-NMR. This type of NMR experiment is best known by
its acronym, COSY. Other types of two-dimensional NMR include J-spectroscopy, exchange
spectroscopy (EXSY), Nuclear Overhauser effect spectroscopy (NOESY), total correlation
spectroscopy (TOCSY), and heteronuclear correlation experiments, such as HSQC, HMQC,
and HMBC. In correlation spectroscopy, emission is centered on the peak of an individual nucleus; if
its magnetic field is correlated with another nucleus by through-bond (COSY, HSQC, etc.) or
through-space (NOE) coupling, a response can also be detected on the frequency of the correlated
nucleus. Two-dimensional NMR spectra provide more information about a molecule than one-
dimensional NMR spectra and are especially useful in determining the structure of a molecule,
particularly for molecules that are too complicated to work with using one-dimensional NMR. The
first two-dimensional experiment.

Solid-state nuclear magnetic resonance

A variety of physical circumstances do not allow molecules to be studied in solution, and at the same
time not by other spectroscopic techniques to an atomic level, either. In solid-phase media, such as
crystals, microcrystalline powders, gels, anisotropic solutions, etc., it is in particular the dipolar
coupling and chemical shift anisotropy that become dominant to the behaviour of the nuclear spin
systems. In conventional solution-state NMR spectroscopy, these additional interactions would lead
to a significant broadening of spectral lines. A variety of techniques allows establishing high-
resolution conditions, that can, at least for 13C spectra, be comparable to solution-state NMR spectra.

Two important concepts for high-resolution solid-state NMR spectroscopy are the limitation of
possible molecular orientation by sample orientation, and the reduction of anisotropic nuclear
magnetic interactions by sample spinning. Of the latter approach, fast spinning around the magic
angle is a very prominent method, when the system comprises spin 1/2 nuclei. Spinning rates of ca.
20 kHz are used, which demands special equipment. A number of intermediate techniques, with
samples of partial alignment or reduced mobility, is currently being used in NMR spectroscopy.

Applications in which solid-state NMR effects occur are often related to structure investigations on
membrane proteins, protein fibrils or all kinds of polymers, and chemical analysis in inorganic
chemistry, but also include "exotic" applications like the plant leaves and fuel cells. For example,
Rahmani et al. studied the effect of pressure and temperature on the bicellar structures' self-
assembly using deuterium NMR spectroscopy.

Biomolecular NMR spectroscopy

Proteins
Much of the innovation within NMR spectroscopy has been within the field of protein
NMR spectroscopy, an important technique in structural biology. A common goal of these
investigations is to obtain high resolution 3-dimensional structures of the protein, similar to what can
be achieved by X-ray crystallography. In contrast to X-ray crystallography, NMR spectroscopy is
usually limited to proteins smaller than 35 kDa, although larger structures have been solved. NMR
spectroscopy is often the only way to obtain high resolution information on partially or
wholly intrinsically unstructured proteins. It is now a common tool for the determination
of Conformation Activity Relationships where the structure before and after interaction with, for
example, a drug candidate is compared to its known biochemical activity. Proteins are orders of
magnitude larger than the small organic molecules discussed earlier in this article, but the basic
NMR techniques and some NMR theory also applies. Because of the much higher number of atoms
present in a protein molecule in comparison with a small organic compound, the basic 1D spectra
become crowded with overlapping signals to an extent where direct spectral analysis becomes
untenable. Therefore, multidimensional (2, 3 or 4D) experiments have been devised to deal with this
problem. To facilitate these experiments, it is desirable to isotopically label the protein with 13C
and 15N because the predominant naturally occurring isotope 12C is not NMR-active and the nuclear
quadrupole moment of the predominant naturally occurring 14N isotope prevents high resolution
information from being obtained from this nitrogen isotope. The most important method used for
structure determination of proteins utilizes NOE experiments to measure distances between atoms
within the molecule. Subsequently, the distances obtained are used to generate a 3D structure of the
molecule by solving a distance geometry problem. NMR can also be used to obtain information on
the dynamics and conformational flexibility of different regions of a protein.

Nucleic acids
Nucleic acid NMR is the use of NMR spectroscopy to obtain information about the structure and
dynamics of polynucleic acids, such as DNA or RNA. As of 2003, nearly half of all known RNA
structures had been determined by NMR spectroscopy.

Nucleic acid and protein NMR spectroscopy are similar but differences exist. Nucleic acids have a
smaller percentage of hydrogen atoms, which are the atoms usually observed in NMR spectroscopy,
and because nucleic acid double helices are stiff and roughly linear, they do not fold back on
themselves to give "long-range" correlations. The types of NMR usually done with nucleic acids
are 1H or proton NMR, C NMR, N NMR, and P NMR. Two-dimensional NMR methods are almost
always used, such as correlation spectroscopy (COSY) and total coherence transfer spectroscopy
(TOCSY) to detect through-bond nuclear couplings, and nuclear Overhauser effect spectroscopy
(NOESY) to detect couplings between nuclei that are close to each other in space.

Parameters taken from the spectrum, mainly NOESY cross-peaks and coupling constants, can be
used to determine local structural features such as glycosidic bond angles, dihedral angles (using
the Karplus equation), and sugar pucker conformations. For large-scale structure, these local
parameters must be supplemented with other structural assumptions or models, because errors add
up as the double helix is traversed, and unlike with proteins, the double helix does not have a
compact interior and does not fold back upon itself. NMR is also useful for investigating nonstandard
geometries such as bent helices, non-Watson–Crick basepairing, and coaxial stacking. It has been
especially useful in probing the structure of natural RNA oligonucleotides, which tend to adopt
complex conformations such as stem-loops and pseudoknots. NMR is also useful for probing the
binding of nucleic acid molecules to other molecules, such as proteins or drugs, by seeing which
resonances are shifted upon binding of the other molecule.

Carbohydrates
Carbohydrate NMR spectroscopy addresses questions on the structure and conformation
of carbohydrates. The analysis of carbohydrates by 1H NMR is challenging due to the limited
variation in functional groups, which leads to 1H resonances concentrated in narrow bands of the
NMR spectrum. In other words, there is poor spectral dispersion. The anomeric proton resonances
are segregated from the others due to fact that the anomeric carbons bear two oxygen atoms. For
smaller carbohydrates, the dispersion of the anomeric proton resonances facilitates the use of 1D
TOCSY experiments to investigate the entire spin systems of individual carbohydrate residues.

Drug Discovery
Knowledge of energy minima and rotational energy barriers of small molecules in solution can be
found using NMR, e.g. looking at free ligand conformational preferences and conformational
dynamics, respectively. This can be used to guide drug design hypotheses, since experimental and
calculated values are comparable. For example, AstraZeneca uses NMR for its oncology research &
development.

NMR spectroscopy and rechargeable batteries

Rechargeable batteries are complex and heterogeneous devices with numerous interfaces, which
are essential as they are at the core of the battery function. The redox reactions used to store and
release energy necessitate the (triple) contact of electrons, redox centres, and ions
(commonly lithium) for charge balance. Side reaction also take place at interfaces and they are
therefore of key importance for the battery lifetime. Two main families of measurements, ex situ and
in situ, can be performed to study the solid interphases in batteries. For ex situ NMR, the part of
interest is extracted from the battery in an argon glovebox and transferred into the NMR sample
holder. Magic Angle Spinning (MAS) is a great tool to record resolved NMR spectra of solids and it is
the main asset of ex-situ NMR for the characterization of the solid components of a battery,
especially for positive paramagnetic electrodes-electrodes containing transition metal ions with
localized unpaired electron such as Co2+, Ni2/3+, Mn4+, Fe2/3+ ...

A 200 MHz NMR instrument with a 4.7 T magnet at CEMHTI-CNRS, Orléans, France.

Ex situ NMR is traditionally performed to study the bulk changes in the solid parts for various state of
charge of the battery and more recently, it was applied to gain insight into the interface components
of Lithium-ion battery, especially the solid electrode-electrolyte interface (SEI) for the anode, the
solid electrolyte reactivity and dynamics and the cathode-electrolyte interface (CEI) for the cathode,
but also for Sodium-ion battery. The liquid electrolyte stability (decomposition products on the
surface) and the plating and stripping of metal (lithium, sodium) on negative electrodes were of
particular interest. The main issue for NMR studies of interphases is the small amount of material so
that the signals for the interphase are often hidden or negligible compared to the intense signals of
the bulkier parts, such as the electrolyte or electrode.

For in situ NMR, the full battery is placed within the NMR magnet and radiofrequency coil for the
measurement. Unfortunately, in situ NMR suffers from lower resolution, so that in situ approaches
concentrate on batteries with materials displaying strong variations in the NMR shifts upon charge-
discharge, or in combination with ex situ studies. In situ NMR is advantageous as it is non-
destructive - the battery does not need a destructive opening for the measurement - and it allows
measuring the spectra for several states of charge on the same battery. Operando
spectroscopy (while the current is flowing for the charge/discharge) enables detecting transient
phases in real-time which is of particular interest in batteries because of the strongly reducing
environment, especially at the negative electrode on top of charge.