Nuclear magnetic resonance (NMR) spectroscopy exploits the magnetic properties of atomic nuclei. It can provide information about the physical and chemical properties, structure, dynamics, and kinetics of biochemical systems. NMR spectroscopy works by aligning atomic nuclei with an external magnetic field and then stimulating them with radiofrequency pulses. This causes the nuclei to absorb and emit radiofrequency radiation at characteristic frequencies. Analyzing these frequencies yields information about the molecule's structure. NMR is widely used to determine the structures of organic molecules and biomolecules like proteins and nucleic acids.

1. Nuclear magnetic resonance
(NMR)
By: Dr. Ashish C Patel
Assistant Professor
Vet College, AAU, Anand

2. • Nuclear magnetic resonance spectroscopy, most commonly known as
NMR spectroscopy, is a research technique that exploits the magnetic
properties of certain atomic nuclei.
• This type of spectroscopy determines the physical and chemical
properties of atoms or the molecules in which they are contained.
• Nuclear magnetic resonance (NMR) spectroscopy is a powerful
technique that can be used to investigate the structure, dynamics, and
chemical kinetics of a wide range of biochemical systems.
• The first NMR derived three dimensional solution structure of a
small protein was determined in 1985 means NMR is about 25
years earlier than X-ray crystallography

3. • NMR spectroscopy can provide information about conformational
dynamics and exchange processes of biomolecules at timescales
ranging from picoseconds to seconds
• Efficient in determining ligand binding and mapping interaction
surfaces of protein/ligand complexes.
• Nowadays, three-dimensional structures can be obtained for proteins
up to 50 kDa molecular weight, and NMR spectra can be recorded
for molecules well above 100 kDa.

4. • This technique relies on the ability of atomic nuclei to behave
like a small magnet and align themselves with an external
magnetic field.
• When irradiated with a radio frequency signal the nuclei in a
molecule can change from being aligned with the magnetic
field to being opposed to it.
• The instrument works on stimulating the “nuclei” of the atoms
to absorb radio waves. The energy frequency at which this
occurs can be measured and is displayed as an NMR spectrum.
• The most common atomic nuclei observed using this
technique are 1
H and 13
C, but also 31
P, 19
F, 29
Si and 77
Se NMR are
available.

5. History
• 1946 Bloch, Purcell introduced about nuclear magnetic
resonance
• 1955 Solomon gave concept about NOE (nuclear Overhauser
effect)
• 1966 Ernst, Anderson introduced Fourier transform NMR
• 1975 Jeener, Ernst gave concept about 2D NMR
• 1985 Wuthrich first solution structure of a small protein
(BPTI) from NOE derived distance restraints
• 1987 3D NMR 13
C, 15
N isotope labeling of recombinant
proteins

6. • Two common types of NMR spectroscopy are used to
characterize organic structure:
– 1
H NMR:- Used to determine the type and number of H
atoms in a molecule
– 13
C NMR:- Used to determine the type of carbon atoms in
the molecule
6

7. • The source of energy in NMR is radio waves which have long
wavelengths having more than 107
nm, and thus low energy
and frequency.
• When low-energy radio waves interact with a molecule, they
can change the nuclear spins of some elements, including 1
H
and 13
C.
7

8. In a magnetic field, there are two energy states for a proton: a lower
energy state with the nucleus aligned in the same direction as Bo, and
a higher energy state in which the nucleus aligned against Bo.
When an external energy source that matches the energy difference
between these two states is applied, energy is absorbed, causing the
nucleus to “spin flip” from one orientation to another.
The energy difference between these two nuclear spin states
corresponds to the low frequency RF region of the electromagnetic
spectrum.
When a charged particle such as a proton spins on its axis, it creates a
magnetic field. Thus, the nucleus can be considered to be a tiny bar
magnet.
Normally, these tiny bar magnets are randomly oriented in space.
However, in the presence of a magnetic field B0, they are oriented
with or against this applied field.
8

9. More nuclei are oriented with the applied field because this
arrangement is lower in energy.
The energy difference between these two states is very small (<0.1
cal).
9

10. • Thus, two variables characterize NMR: an applied magnetic
field B0, the strength of which is measured in tesla (T), and
the frequency n of radiation used for resonance, measured
in hertz (Hz), or megahertz (MHz).
 The energy difference between two nuclear spin states (v)
needed for resonance and the applied magnetic field strength
(B0) are proportionally related:
 The stronger the magnetic field, the larger energy difference
between two nuclear spin states (v) and higher the ν needed for
the resonance.10
A nucleus is in resonance when it absorbs RF radiation and
“spin flips” to a higher energy state.
A nucleus is in resonance when it absorbs RF radiation and
“spin flips” to a higher energy state.
ν α BO
ν α BO

11. • Both liquid and solid type of samples can be used in NMR
spectroscopy.
• For liquid sample, conventional solution-state NMR spectroscopy is
used for analysing where as for solid type sample, solid-state
spectroscopy NMR is used.
• In solid-phase media, samples like crystals, microcrystalline
powders, gels, anisotropic solutions, proteins, protein fibrils or all
kinds of polymers etc. can be used.
• In liquid phase, different types of liquid solutions, nucleic acid,
protein, carbohydrates etc. can be used.
11

12. 12
Electromagnet

13. 1.Sample holder :- Glass tube with 8.5 cm long,0.3 cm in diameter
2.Permanent magnet :- It provides homogeneous magnetic field at 60-100
MHZ
3.Magnetic coils :- These coils induce magnetic field when current flows
through them.
4.Sweep generator :- To produce the equal amount of magnetic field pass
through the sample
Radiofrequency
Transmitter
Sweep
Generator
Radiofrequency
Amplifier
Audio
Amplifier
Detector
Oscilloscop
and / or
Recorder

14. • Radio frequency transmitter:- A radio coil transmitter that
produces a short powerful pulse of radio waves
• Radiofrequency :- A radio receiver coil that detects Receiver
radio frequencies emitted as nuclei relax to a lower energy
level
• Readout system :- A computer that analyses and record the
data

15. • All subatomic particles (neutrons, protons, electrons) have the
fundamental property of spin. This spin corresponds to a small
magnetic moment.
• In the absence of a magnetic field the moments are randomly
aligned. When a static magnetic field, Bo is applied this field
acts as a turning force that aligns the nuclear spin axis of
magnetic nuclei with the direction of the applied field
• This equilibrium alignment can be changed to an excited state
by applying radio frequency (RF) pulses.
• When the nuclei revert to the equilibrium they emit RF
radiation that can be detected
Principles of nuclear magnetic
resonance

16. • The nuclei of many elemental isotopes have a characteristic spin
(I). Some nuclei have integral spins (e.g. I = 1, 2, 3 ....), some
have fractional spins (e.g. I = 1/2, 3/2, 5/2 ....), and a few have
no spin, I = 0 (e.g. 12
C, 16
O, 32
S, ....).
• Isotopes of particular interest and use are 1
H, 13
C, 19
F and 31
P, all of
which have I = 1/2.
• A spinning charge generates a
magnetic field and resulting spin-
magnet has a magnetic moment (μ)
proportional to the spin.
• In the presence of an external
magnetic field (B0), two spin states
exist, +1/2 and -1/2.

17. • The difference in energy between the two spin states is
dependent on the external magnetic field strength, and is
always very small.
• Strong magnetic fields are necessary for nmr spectroscopy.
• Modern nmr spectrometers use powerful magnets having
fields of 1 to 20. Even with these high fields, the energy
difference between the two spin states is less than 0.1
cal/mole.

18. • For nmr purposes, this small energy difference (ΔE) is usually
given as a frequency in units of MHz (106
Hz), ranging from 20
to 900 Mz, depending on the magnetic field strength and the
specific nucleus being studied.
• For spin 1/2 nuclei the energy difference between the two spin
states at a given magnetic field strength will be proportional to
their magnetic moments.
• In order to induce nmr, a oscillatory magnetic field has to be
applied at the frequency which corresponds to the separation
(ΔΕ) of the two spin energy levels.

19. How does it work
• To get the nuclei in a molecule to all align in the same
direction, a very strong magnetic field is generated using a
superconducting electromagnet, which requires very low
temperatures to function.
• The coils of the magnet are surrounded by liquid helium
(-269°C), which is prevented from boiling off too quickly by a
surrounding layer of liquid nitrogen (-196°C). These coolants
are all contained in double-layer steel with a vacuum between
the layers, to provide insulation just like a thermos.
• There is a narrow hole through the middle of the magnet, and
the sample tube and radio frequency coils ("probe”) are
located there.

20. • A solution of the sample in a uniform 5 mm glass tube is oriented between the
poles of a powerful magnet, and is spun to average any magnetic field variations.
• Radio frequency radiation of appropriate energy is broadcast into the sample from
an antenna coil (colored red). A receiver coil surrounds the sample tube, and
emission of absorbed rf energy is monitored by dedicated electronic devices and a
computer.
Radio Frequency
Transmitter
Magnet
Pole
Sweep Generator
Sweep
Coils
Sweep
Coils
Spinning
Sample
tube
Magnet
Pole
Radio Frequency
Receiver & Amplifier
Control
Console and
Recorder

21. • An nmr spectrum is acquired by varying the magnetic field
over a small range while observing the rf signal from the
sample. An equally effective technique is to vary the
frequency of the rf radiation while holding the external field
constant.

22. Chemical shifts
• The exact resonance frequency depends on the chemical
environment of each spin, such that for example the NMR
spectrum of a protein will show NMR signals with slightly
different frequencies. These differences are called chemical
shifts.
• The first step of a structure determination by NMR consists of
assigning the chemical shifts of all the atoms/spins of the
molecule which are observed in an NMR spectrum.
• The resonance frequencies are called chemical shifts and are
measured in parts per million (ppm) in order to have chemical
shift values independent of the static magnetic field strength.

23. • Backbone amide protons HN
in a protein resonate around 8
ppm, while Hα
spins have resonance frequencies between 3.5-
5.5 ppm.

24. Factors affecting chemical shift:
• Electronegative groups
• Magnetic anisotropy of π-systems
• Hydrogen bonding
• Electronegative groups:- Electronegative groups attached to the C-H
system decrease the electron density around the protons, and there is
less shielding (i.e.deshielding) and chemical shift increases
• Magnetic anisotropy of π-systems:- The word "anisotropic" means
"non-uniform". So magnetic anisotropy means that there is a "non-
uniform magnetic field".
• Electrons in π systems (e.g. aromatics, alkenes, alkynes, carbonyls etc.)
interact with the applied field which induces a magnetic field that
causes the anisotropy. It causes both shielding and deshielding of
protons. Example:-Benzene Hydrogen bonding:- Protons that are
involved in hydrogen bonding are typically change the chemical shift
values. The more hydrogen bonding, the more proton is deshielded and
chemical shift value is higher.

25. Proton NMR
• The most common form of NMR is based on the hydrogen-1 (1H), nucleus or
proton. It can give information about the structure of any molecule containing
hydrogen atoms.

26. Nuclear overhauser effect
• NOE is the transfer of nuclear spin polarization from one spin to
another spin via cross-talk between different spins (normally protons)
in a molecule and it depends on the through space distance between
these spins.
• The local field at one nucleus is affected by the presence of another
nucleus. The result is a mutual modulation of resonance frequencies.
• NOEs are typically only observed between protons which are
separated by less than 5-6 Å.
• NOE is related to the three-dimensional structure of a molecule. For
interproton distances > 5 Å, the NOE is too small and not observable.

27. J coupling constants
• Provide information about dihedral angles, and thus can define
the peptide backbone and side chain conformations.
• Mediated through chemical bonds connecting two spins. The
energy levels of each spin are slightly altered depending on
the spin state of a scalar coupled spin (α or β).

28. Structure determination by
solution NMR

29. NMR spectrum
• An NMR spectrum appears as a series of vertical peaks/signals
distributed along the x-axis of the spectrum.
• Each of these signals corresponds to an atom within the
molecule being observed
• The position of each signal in the spectrum gives information
about the local structural environment of the atom producing
the signal.
• As we move towards bigger molecules with more and more
atoms, the 1D spectra become very complex, and 2D and 3D
spectroscopy becomes important in understanding the
relationships and interactions between different atoms in the
molecule.

30. • The information contained in 1D spectra can be expanded in a
second (frequency) dimension - 2D NMR
• In a 1D experiment a resonance (line) is identified by a single
frequency: NH(f1nh)
• In 2D spectra, a resonance (cross-peak) is identified by two
different frequencies: NH (f1nh, f2ha), NH (f1nh, f2ha)
• Usually, the second frequency depends on how the NMR
experiment is designed.

31. Resonance assignment
• The crosspeaks in NOE spectra cannot be interpreted without knowledge
of the frequencies of the different nuclei
• The frequencies can be obtained from information contained in COSY
(correlation spectroscopy) spectra
• The process of determining the frequencies of the nuclei in a molecule is
called resonance assignment (and can be lengthy...)
• Two-dimensional COSY NMR experiments give correlation signals that
correspond to pairs of hydrogen atoms which are connected through chemical
bonds.
• COSY spectra show frequency correlations between nuclei that are connected
by chemical bonds
• Since the different amino acids have a different chemical structure they give
rise to different patterns in COSY spectra. This information can be used to
determine the frequencies of all nuclei in the molecule. This process is called
resonance assignment
• Modern assignment techniques also use information from COSY experiments
with 13C and 15N nuclei

32. • NOE Spectroscopy experiments
give signals that correspond to
hydrogen atoms which are close
together in space (< 5A), even
though they may be far apart in the
amino acid sequence.
• Structures can be derived from a
collection of such signals which
define distance constraints between
a number of hydrogen atoms along
the polypeptide chain.
Example: short distance (< 5 A, NOE)
correlations between hydrogen atoms in a
helix

33. Multi-Dimensional NMR:
Built on the 3D Principle

34. • NMR is used in biology to study the Biofluids, cells, organs and
macromolecules such as Nucleic acids (DNA, RNA), carbohydrates
Proteins and peptides and also Labeling studies in biochemistry.
• NMR is used in physics and physical chemistry to study High pressure
diffusion ,liquid crystals, Membranes.
• 3D structure determination of proteins, nucleic acids, protein/DNA
complexes, ...)
• NMR is used in pharmaceutical science to study Pharmaceuticals and
Drug metabolism.
• NMR is used in chemistry to determine the Enantiomeric purity.
Elucidate Chemical structure of organic and inorganic compounds.
• 1
H widely used for structure elucidation.
Inorganic solids- Inorganic compounds are investigated by solid state
1
H-NMR.eg CaSO4 H2O.⋅
Organic solids- Solid-state 1
H NMR constitutes a powerful approach to
investigate the hydrogen-bonding and ionization states of small organic
compounds.
Applications of NMR

35. • Direct correlation with hydrogen-bonding lengths could be
demonstrated, e.g. for amino acid carboxyl groups.
• Polymers and rubbers- Examine hydrogen bonding and acidity.
• In vivo NMR studies- concerned with 1H NMR of human brain. Many
studies are concerned with altered levels of metabolites in various brain
diseases.
• To determine the spatial distribution of any given metabolite detected
spectroscopically IS (image selected in vivo spectroscopy).
• MRI is specialist application of multi dimensional Fourier
transformation NMR for Anatomical imaging, for measuring
physiological functions, for flow measurements and angiography, for
tissue perfusion studies and also for tumors.

36. Limits
• Molecular weight limits for protein structure calculation (monomer):
5-15 kDa: routine
15-20 kDa: usually feasible
20-30 kDa: long term project
40-50 kDa: in the next future?
• Molecular weight limits for peptide/protein, protein/protein interactions
(MW of the AB complex, A < 10 kDa):
20-30 kDa: routine
30-50 kDa: feasible
50-100 kDa: in the next future

37. Advantages of NMR Limitation of NMR
Obtain angles, distances, coupling constants,
chemical shifts, rate constants etc. These are
really molecular parameters which could be
examined more with computers and molecular
modeling procedures.
This is good for the more accurate determination
of the structure, but not for the availability of
higher molecular masses
With a suitable computer apparatus we can
calculate the whole 3D structure
The resolving power of NMR is less than some
other type of experiments (e.g.: X-ray
crystallography) since the information got from
the same material is much more complex
There are lots of possibilities to collect different
data-sets from different types of experiments for
the ability to resolve the uncertanities of one
type of measurements
The highest molecular mass which was examined
successfully is just a 64kDa protein-complex
This method is capable to lead us for the
observation of the chemical kinetics
There are lots of cases when from a given data-
set - a given type of experiment – we may
predict two or more possible conformations, too
We can investigate the influence of the dielectric
constant, the polarity and any other properties of
the solvent or some added material
The cost of the experimental implementation is
increasing with the higher strength and the
complexity of the determination

38. Disadvantages
1) Sensitivity
• The greatest disadvantage of NMR spectroscopy and imaging compared with
other modalities is the intrinsic insensitivity of the methods. The signal that
can be generated in the
• NMR experiment is small and, for practical purposes, most strongly coupled
with the concentration of the nuclei in the sample.
• For example, the human body is composed of -70% water, and thus a
relatively large signal can be obtained from the 1
H nucleus in water that is
effectively at a concentration in the tens of molar range.
• Thus, it is possible to measure signals from cubes (voxels) of tissue as small as
= 0.3mm on a side from the human brain, generating the high-quality.
• The NMR signals from water will always be detectable at resolutions
approximately two orders of magnitude greater than those of other NMR-
sensitive nuclei.
• Thus, compounds present in submillimolar and certainly micromolar
concentrations cannot practically be detected directly in tissues.
• As a result, the sample size generally dictates the choice of magnet and field
strength; thus, the smaller the sample, the more sensitive the experiments.

39. 2) Working in a High-Magnetic-Field Environment
• An inevitable consequence of carrying out NMR investigations is the need to
work in a high-magnetic-field environment.
• No known intrinsic risks are associated with high magnetic fields; however, the
presence of the magnetic field can affect equipment routinely used in animal
research.
• For example, electronic monitors and computer-controlled devices may
function improperly or not at all.
• Due to the nature of the forces involved, the result can be a scalpel, a pair of
scissors, or even a gas cylinder becoming a flying object.
• Instruments that can be obtained that are non ferromagnetic, thus reducing the
potential difficulties associated with working in a high-magnetic-field
environment.
• Now a days, a steel passive shield or an active shield may be placed around the
magnet to reduce the magnet fringe fields and minimize the risks.
• This reduction can be particularly important when the space available to site
the instrument is limited.

40. 3) Motion Sensitivity
• Most MR techniques are motion sensitive. This sensitivity leads to
signal distortions that are visually most evident in artifacts on images
or more subtly in quantitative measurements.
• Some MR techniques such as functional MRI (fMRI1) are particularly
sensitive to motion artifact, thus great care must be taken not to
minimize the distorting effects of motion and thus minimize
misinterpretation of data.
• In animal studies, anesthesia is usually essential to avoid gross
movement of the animal during the study.
• Cardiac gating may be necessary even when imaging other organs
such as the brain because of motion caused by the pulsatile blood
flow. In some cases, such as lung imaging, it may also be necessary to
gate to respiratory motion or, alternatively, the subject may be
controlled via mechanical ventilation.

41. Examples/ uses of NMR Spectroscopy
• Several different NMR-sensitive nuclei can be used in the study of
biological systems, and the most common are 31
P, 1
H, 13
C, 23
Na, and 19
F.
• 31
P, 1
H, and 13
C-NMR spectroscopy are typically used to investigate
cellular metabolism and bioenergetics.
• Whereas 23
Na NMR studies usually focus on issues related to ion
transport and regulation of ion pumps. Fluorine does not occur naturally
in biological systems.
• However, it is a very sensitive NMR nucleus. Therefore, fluorine-
labeled compounds can be introduced into cells and used as an indicator
of a cellular process such as calcium concentration.
• 19
F-NMR spectroscopy has also been used to follow the metabolism of
drugs, such as 5-fluorouracil.

42. NMR and X-ray crystallography are
complementary
• Molecules are studied in solution.
• Protein folding studies can be done by monitoring NMR spectra
• Denatured states of a biomolecule, folding intermediates and even
transition states can be characterized
• Conformational or chemical exchange, internal mobility and
dynamics at timescales ranging from picoseconds to seconds
• Very efficient in mapping interactions with other molecules
• Upper weight limit for NMR is ~ 50 kDa