NMR spectroscopy exploits the magnetic properties of atomic nuclei to characterize organic molecules. It works by applying a strong magnetic field to align nuclear spins, and then applying a radiofrequency pulse to induce transitions between spin states. This allows identification of carbon-hydrogen frameworks within molecules. The NMR spectrum provides information on chemical environments and molecular structure through properties like chemical shift, spin-spin splitting, integration, and coupling constants. Developments include 2D NMR, deuterium labeling, and Fourier transform NMR for improved resolution and sensitivity.

1. NMR Spectroscopy
Presented by
Shaheda Parveen
PhD Scholar
Department of Biotechnology
Jamia Milia Islamia

2. Introduction
• NMR was first experimentally observed by Bloch and Purcell in 1946 (received
Nobel Prize in 1952)
• Nuclear magnetic resonance spectroscopy (NMR) is a powerful analytical
technique used to characterize organic molecules by identifying carbon-hydrogen
frameworks within molecules.
• exploits the magnetic properties of certain atomic nuclei.
• It is a branch of spectroscopy in which radio frequency waves induce transitions
between magnetic energy levels of nuclei of a molecule.
• NMR is concerned with change in the direction of spin orientation as the result of
the absorption of radiofrequency radiation.

3. Magnetic phenomena
• Magnetism arises from the motion of charged particles.
• The nuclei of atoms are composed of protons and neutrons. Like electrons, these
particle also have the properties to spin on their own axis and each of them
possesses angular momentum 1/2(h/2π)
• The net resultant of the angular momentum of all nuclear particles is called nuclear
spin
• Each spin-active nucleus has a number of spins defined by its spin quantum
number, I.
• The number of Spin states = 2I + 1

4. Principle of NMR
• The NMR is mostly consult with
nucleus spin quantum no. (I)= ½ .
• The proton having a I = ½ when
place in external magnetic field
(Bo) it’s start to spin around the
nuclear axis and generate another
magnetic field.
• According to quantum mechanics
there are 2I + 1 so two spin stage
+ ½ and - ½ for the proton.

5. If a nucleus having a magnetic moment is introduced into a magnetic field , B0, the
two energy levels become separate corresponding to mI = -1/2 and mI = +1/2
For a nucleus with I = 1/2, the energies E1 and E2 for the two states with mI = +1/2
and mI = -1/2 , respectively, are
E1 = -1/2 [ γ h / 2π ] B0
E2 = + 1/2 [ γ h / 2π ] B0
Gyromagnetic ratio, γ, is a constant for each nucleus (26,753 s-1
gauss-1
for H).
relationship:
ν = E2 – E1 / h
ν = 1/2 [ γ h / 2π ] B0 + 1/2 [ γ h / 2π] B0 / h ( or )
ν= γ / 2πB0
This is the Larmor equation

6. From Larmor equation
The frequency of absorbed electromagnetic radiation is
proportional to the energy difference between two
nuclear spin states which is proportional to the applied
magnetic field
Magnetic nuclei are in resonance with external
magnetic field if they absorb energy and “spin-flip”
from low energy state (+1/2) to high energy state (-1/2)

7. Larmor frequency
• When a spin-active hydrogen atom is placed in a strong magnetic field it begins to
precess with larmor frequency.
• May be defined as revolutions per second made by the magnetic moment vector of
the nucleus around the external magnetic field Bo
• Directly proportional to the strength of applied magnetic field.
• Magnetic nuclei of different atoms have different characteristic precessional
frequency.
• according to Larmor precessional theory
ω = γB0 ………………
where, ω= 2 πV, Larmor precessional frequency.

8. Relaxation Time
• After a certain time-span, the spins will return from the higher to the lower energy
level, a process that is known as relaxation.
• There are two principle modes of relaxation,
Longitudinal relaxation /spin lattice:- when energy released during the transition of
a nuclear spin from the higher to the lower energy state, it can be emitted as heat
into the environment and is called spin– lattice relaxation. This process termed as
T1.
Transverse relaxation /spin spin:- The transverse magnetization of the nuclei is also
subject to change over time, due to interactions between different nuclei.
Here, An excited nucleus may transfer its energy to an unexcited nucleus of a similar
molecules that is nearby. There is no net change in energy of the system, but the
length of the time that one nucleus stays excited is shortened. This process, which
is called transverse relaxation T2.

9. Types of NMR
Two common types of NMR spectroscopy are used to characterize organic structure:
• 1
H NMR
• 13
C NMR
Types of samples
• Both liquid and solid
• In solid samples, the number of spin–spin interactions is greatly enhanced due to
intermolecular interactions. As a result, the resonance signals broaden significantly.
However, high-resolution spectra can be obtained by spinning the solid sample at
an angle of 54.7˚ (magic angle spinning)
• Usually, deuterated solvents such as CDCl3 are used for sample preparation of
organic compounds.
• For peptides and proteins , solvent D2O

10. Interpreting Proton NMR Spectra
TYPES OF INFORMATION FROM THE NMR SPECTRUM
1. Different type of hydrogen gives a peak or group of peaks (multiplet).
2. The integral gives the relative numbers of each type of hydrogen.
3. The chemical shift or position of a signal (δ, ppm) gives a clue as to the type of
hydrogen generating the peak (alkane, alkene, benzene, aldehyde, etc.)
4. Spin-spin splitting gives the number of hydrogens on adjacent carbons.
5. The coupling constant ‘J’ also gives information about the arrangement of the
atoms involved.

11. Chemical shift (δ)
• The molecular environment
of a proton governs the value
of the applied external field
at which the nucleus
resonates, called chemical
shift.
• TMS at 0.5% concentration
is used as internal standard.
• Rather than measure the
exact resonance position of a
peak, we measure how far
downfield it is shifted from
TMS.
δ = shift in Hz
spectrometer frequency in MHz
τ (tau) = 10 - δ

12. Shielding and deshielding

13. Chemical shift depends on electronegativity
IT IS USUALLY SUFFICIENT TO KNOW WHAT TYPES OF HYDROGENS COME IN
SELECTED AREAS OF THE NMR CHART

14. Approximate chemical shift ranges (ppm) for
selected types of protons

15. Number of signals
• A set of protons of identical environments are known as equivalent protons while
the protons with different environments are known as non-equivalent protons.
• The number of signals in a NMR spectrum tells us how many kinds of protons are
present in a given molecule.
• Shift reagents are used in case of overlapping peaks.
Acetone –(1) Benzene – (1) Methyl acetate- (2) Ethyl Benzene-
(3)
Propane 2-ol – (3)

16. Integration of a peak
• process called integration
• The area under a peak is proportional to the number of hydrogens that generate the
peak.
• The integral line rises an amount proportional to the number of H in each peak

17. Integration of a peak

18. Spin-spin splitting
• Often a group of hydrogens will appear as a
multiplet rather than as a single peak because of
interaction with neighboring hydrogen.
• The sub peaks are due to spin-spin splitting and
are predicted by the n+1 rule
• Each type of proton senses the number of
equivalent protons (n) on the carbon atom(s) next
to the one to which it is bonded, and its
resonance peak is split into (n+1) components.
• Protons that are equivalent by symmetry usually
do not split one another
Multiplet
Singlet
Quintet
Doublet
Septet
Triplet
Octet
Quartet
Nonet
n +1= 3
Triplet
n +1= 2
Duplet

19. NMR Spectrum of Bromoethane
Br CH2CH3
Triplet
Quartet

20. NMR Spectrum of 2-methyl-1-pentene
e,Singlet
a e
d
b
d,Triplet
a,Triplet
b,Sexlet
c,Singlet
2-methyl-1-pentene
c

21. NMR Spectrum of α-chloro-p-xylene
c
a, singlet
c, multiplet
b, singlet
α-chloro-p-xylene
a
b

22. NMR Spectrum of Phenacetin (C10H13NO2)
ba c e
d
a
b
c
d
e

23. Coupling Constant ‘J’
• The coupling constant is the distance J (in Hz) between the peaks in a simple
multiplet.
• J is a measure of the amount of interaction between the two sets of hydrogens
creating the multiplet.

24. Isotope exchange
• Deuterium (2H or D), has been used extensively in proton NMR spectroscopy for two
reasons. First it is easily introduced into a molecule. Second, the presence of deuterium
in a molecule is not detected in the proton NMR spectrum.
• Deuterium has a much smaller magnetic dipole moment than hydrogen & therefore, it
absorbs at different field strengths.
• In case of ethylbromide the deuterium replaces the methyl hydrogens & the following
changes occurs.

25. 13
C NMR Spectroscopy
• Carbon-13: only carbon isotope with a nuclear spin
• Natural abundance 1.1% of C’s in molecules
• Sample is thus very dilute in this isotope
• Sample is measured using repeated accumulation of data and averaging of signals,
incorporating pulse and the operation of Fourier transform (FTNMR).
• All signals are obtained simultaneously using a broad pulse of energy and resonance recorded.
• Multiplicities in these spectra cannot be observed
13C NMR spectra of 1-pentanol,CH3CH2CH2CH2CH2OH

26. The Pulsed Fourier Transform (ft )
• An alternative approach, uses a powerful but short burst of energy called a pulse that excites
all of the magnetic nuclei in the molecule simultaneously and all the signals are collected at
the same time with a computer.
• because the measured signal in NMR is the re-emission of energy as the nuclei return from
their high-energy into their low-energy states, the recorded radiation will decay with time, as
fewer and fewer nuclei will return to the ground state. The signal measured is thus called the
free induction decay (FID).
• FT-NMR can be obtained with less than 0.5 mg of compound Pulsed FT-NMR is therefore
especially suitable for the examination of nuclei that are magnetic or very dilute samples.

27. 2-D NMR
• Gives data plotted in a space defined by two frequency rather than one
• show chemical shifts on both axes
• Suitable for more complex and large molecules

28. Instrumentation

29. • The sample (2-10 mg ) is dissolved in a solvent containing no interfering protons
usually CCl4 or CdCl3 0.5 ml and a small amount of TMS is added to serve as an
internal reference.
• The sample cell is a small cylindrical glass tube that is suspended in the gap
between the faces of the pole pieces of the magnet. The sample cell is rotated
around its axis to ensure that all parts of the solution experience a relatively
uniform B
• Also in the magnetic gap, the radio frequency oscillator coil is installed
perpendicular (90˚) to the applied magnetic field.
• This coil supplies the electromagnetic energy used to change the spin orientations
of the protons.
• Detector coil is arranged perpendicular to the RF oscillator coil. As the magnetic
field strength is increased, the precessional frequencies of all the nucleus increases
• As the magnetic field strength is increased linearly, a pen travels from left to the
right on a recording chart.
• As each chemically distinct type of proton comes into resonance, it is record as a
peak on the chart..
Working

30. Comparison between 13
C and 1
H NMR

31. Application of NMR
• Molecular structure determination
• Solution structure of proteins and peptides The structures of proteins up to a mass
of about 50 kDa can be determined with NMR spectroscopy.
• Magnetic resonance imaging
• Protein folding The most powerful tool for determining the residual structures of
unfolded proteins and the structures of folding intermediates.
• Material science A powerful tool in the research of polymer chemistry and physics.

32. Thanking you