2. Contents
• Introduction
• History of NMR Spectroscopy
• Principle of NMR Spectroscopy
• Theory of NMR Spectroscopy
• Instrumentation of NMR Spectroscopy
• Various Aspects of NMR Spectroscopy
• Carbon-13 NMR
• Applications of NMR spectroscopy
3. Introduction
• Nuclear magnetic resonance is a spectroscopy technique which
is based on the absorbance of electromagnetic radiation in a
radiofrequency region by the nuclei of the atoms.
• Two common types of NMR spectroscopy are used to
characterize organic structure: 1H NMR is used to determine
the type and number of H atoms in a molecule; 13C NMR is
used to determine the type of carbon atoms in the molecule.
• NMR is the most powerful tool available for organic structure
determination.
• It is used to study a wide variety of nuclei:
• 1H
• 13C
• 15N
• 19F
• 31P
4. NMR History
• 1937 Rabi’s prediction and observation of nuclear magnetic resonance
• 1945 First NMR of solution (Bloch et al for H2O) and solids (Purcell et
al for parafin)!
• 1953 Overhauser NOE (nuclear Overhauser effect)
• 1966 Ernst, Anderson Fourier transform NMR
• 1975 Jeener, Ernst 2D NMR
• 1980 NMR protein structure by Wuthrich
• 1990 3D and 1H/15N/13C Triple resonance
• 1997 Ultra high field (~800 MHz) & TROSY(MW 100K)
5. Continuation of NMR History
Nobel prizes
1944 Physics Rabi (Columbia)
1991 Chemistry Ernst (ETH)
"for his resonance
method for recording
the magnetic
properties of atomic
nuclei"
1952 Physics Bloch (Stanford),
Purcell (Harvard)
"for their development
of new methods for
nuclear magnetic
precision measurements
and discoveries in
connection therewith"
"for his contributions to
the development of the
methodology of high
resolution nuclear
magnetic resonance
(NMR) spectroscopy"
6. Continuation of NMR History
2002 Chemistry Wüthrich (ETH)
2003 Medicine Lauterbur (University of Illinois in
Urbana ), Mansfield (University of Nottingham)
"for his development of nuclear magnetic
resonance spectroscopy for determining the
three-dimensional structure of biological
macromolecules in solution"
"for their discoveries concerning
magnetic resonance imaging"
7. Principle of NMR spectroscopy
• When a substance is placed in a magnetic field of constant
strength and then obtain a spectrum, by passing the
radiation of steadily changing frequency through the
substance and observe the frequency at which radiation is
absorbed .
• In practice, however , it has been found more convenient
to keep the radiation frequency constant and to vary the
strength of magnetic field ; at some value of field strength,
the energy required to flip the proton matches the energy
of the radiation ; absorption occurs and signal is observed.
Such a spectrum is called as the nuclear magnetic
resonance spectrum.
9. Theory of NMR Spectroscopy
• A nucleus with an odd atomic number or an odd mass
number has a nuclear spin.
• The spinning charged nucleus generates a magnetic field
=>
10. Theory of NMR Spectroscopy
• When placed in an external field, spinning protons act like
bar magnets.
11. Theory of NMR Spectroscopy
• The magnetic fields of the spinning nuclei will align either
with the external field, or against the field.
• The nucleus will have two spin states, one is called as the
state (with lower energy) and other is called as the β spin
state (with higher energy state).
• A photon with the right amount of energy can be absorbed
and cause the spinning proton to flip.
12. Theory of NMR Spectroscopy
• When the energy of the photon matches the energy
difference between the two spin states , an absorption of
energy occurs. We call that phenomenon Resonance
• Energy difference is proportional to the magnetic field
strength.
• E = h = h B0
2
• Gyromagnetic ratio, , is a constant for each nucleus
(26,753 s-1gauss-1 for H).
• In a 14,092 gauss field, a 60 MHz photon is required to flip
a proton.
13. Instrumentation of NMR SPECTROSCOPY
An NMR machine consists of:
• Sample holder
• Permanent magnet
• Magnetic coils
• Sweep generator
• Radio frequency Transmitter
• Radio frequency Receiver
15. Instrumentation of NMR SPECTROSCOPY
• Sample Holder : Glass tube with 8.5cm long, 0.3cm in diameter.
• Permanent magnet : It provides homogenous magnetic field. Either a
permanent magnet or electromagnet can be employed to supply
magnetic field. Currently superconducting magnets cooled in liquid
helium are being used in instrument which require high magnetic
strength.
• Magnetic coils: These coils induce the magnetic field , when current
flow through them.
• Sweep Generator: It produce the equal amount of magnetic field that
pass through the sample.
16. Instrumentation of NMR SPECTROSCOPY
• Radiofrequency transmitter: A radio transmitter coil that produce a
powerful radio waves.
• Radiofrequency detector: The flipping of nuclei as a result of
irradiation induces a voltage in receiving coil.
• Recorder : the voltage from the receiving coil is amplified and
observed in a recorder. The peaks of an NMR spectrum are result of
plotting intensity of absorption Vs. frequency of strength.
17. Solvents used in NMR
Solvent used in NMR spectroscopy should have the following
properties:
Non viscous.
Should dissolve analyte.
Should not absorb within spectral range of analysis.
All solvents used in NMR must be aprotic that is they should not possess proton.
• Chloroform-d (CDCl3) is the most common solvent for NMR
measurements
• Other deuterium labeled compounds, such as deuterium oxide
(D2O), benzene-d6 (C6D6), acetone-d6 (CD3COCD3) and DMSO-d6
(hexaduetereo dimethylsulfoxide) are also available for use as NMR
solvents.
• DMF (dimethylformide), DMSO, cyclopropane, dimethyl ether can
also be used.
18. NMR spectrum
• Various aspects of NMR spectrum is helpful in determining
the structure of the molecules.
a) The number of signal
b) The position of the signal
c) The intensities of the signal
d) The splitting of a signal
19. The number of signal
• The number of signals in the NMR spectrum shows how many
different kinds of protons are present in the molecule.
20. Position of the signal (chemical shift)
• Position of the signal indicate the kinds of protons. They may be
aromatic, aliphatic, primary, secondary, tertiary ,benzylic , vinylic, or
to other atoms or groups.
• When a molecule is placed in a magnetic field, its electron are also
caused to circulate and, in circulating, they generate the secondary
magnetic field; induced magnetic field, which may either i) opposes
the applied field ; the field felt by the proton is thus diminished, and
the proton is said to be shielded or ii) reinforces the applied field,
then the field felt by the proton is said to be desheilded.
• Compared with the naked proton, a shielded proton requires a
higher applied field strength and a deshielded proton requires a
lower applied field strength to provide the particular effective field
strength at which absorption occurs.
22. Cont….
• Shielding thus shifts the absorption upfield , deshielding shifts the
absorption downfield.
• Such shifts in the position of NMR absorptions, arising from shielding
and deshielding by electrons, are called as the chemical shift.
• So, a chemical shift is defined as the difference in parts per million
(ppm) between the resonance frequency of the observed photon
and the Tetra methylsilane hydrogens.
• TMS is the most common refrence compound in NMR . It is set at
δ = 0 ppm.
Chemical shift = frequency of signal –frequency of reference x 106
Spectrometer frequency
23. Cont….
NMR absorptions generally appear as sharp peaks.
Increasing chemical shift is plotted from left to right.
Most protons absorb between 0-10 ppm.
The terms “upfield” and “downfield” describe the relative location of
peaks. Upfield means to the right. Downfield means to the left.
24. Cont…..
• Measured in parts per million.
• Ratio of shift downfield from TMS (Hz) to total
spectrometer frequency (Hz).
• Same value for 60, 100, or 300 MHz machine.
• Called the delta scale.
25. Cont….
• Mostly chemical shifts have value between 0 and 10.
• A small delta value represents a small downfield shield, and a large
delta value represents a large downfield shift.
26. Tetramethylsilane
• TMS is added to the sample.
• Since silicon is less electronegative , TMS protons are highly shielded.
Signal defined as zero.
• Organic protons absorb downfield (to the left) of the TMS signal.
Si
CH3
CH3
CH3
H3C
27. Factors affecting the chemical shift
Chemical shifts is influenced by the following factors;
ELECTRONEGATIVE GROUPS:
• More electronegative atoms Deshields more and give larger shift
values.
• Effect decreases with distance.
• Additional electronegative atoms cause increase in chemical shift.
• Attachment of electronegative atom cause a downward shift.
Anisotropic effect:
• The word anisotropic means “non uniform”. So magnetic anisotropy
means that there is a non uniform magnetic field.
• Electron in 𝜋 system (aromatics, alkenes, alkynes, carbonyl etc)
interact with the applied field that cause the anisotropy.
• It cause both shielding and deshielding.
28. Factors affecting the chemical shift
Hydrogen bonding:
Protons that are involved in chemical bonding are typically
change the chemical shift values.
The more hydrogen bonding is , the more proton is
deshielded and chemical shift value is higher.
29. Intensity of the signal
• The area under each peak is proportional to the number of protons.
• Shown by integral trace.
30. Spin-spin coupling
• The effect of a spin of a proton on adjacent spinning proton
(nonequivalent) resulting in splitting of its absorption signal is called
as the spin-spin coupling.
• Nonequivalent protons on adjacent carbons have magnetic fields that
may align with or oppose the external field.
• This magnetic coupling causes the proton to absorb slightly downfield
when the external field is reinforced and slightly upfield when the
external field is opposed.
• All possibilities exist, so signal is split.
32. Spin-spin coupling
Example: Splitting Patterns in 1,1,2-tribromoethane
The methine (-CH-) hydrogen can assume two magnetic spin orientations, with or against the external field. As a result the peak
for the adjacent methylene group is split into two lines of equal intensity,a doublet.
The methylene (-CH2-) hydrogen atoms can again assume one of two magnetic spin orientations, with or against the external
field. However, in this case these are two H atoms that are identical so there are three possible combinations of their two spins.
The first is both are spin down, the next is both spin up (shown on the right). There are two possible ways for the 1 spin up and
1 spin down combination depending on which specific H atom is spin up or down (shown in the center). The combination
produces a triplet with relative intensities of 1:2:1 for the adjacent methine group.
35. 35
The N + 1 Rule
If a signal is split by N equivalent protons,
it is split into N + 1 peaks.
=>
36. 36
Range of Magnetic
Coupling
• Equivalent protons do not split each other.
• Protons bonded to the same carbon will split each other
only if they are not equivalent.
• Protons on adjacent carbons normally will couple.
• Protons separated by four or more bonds will not couple.
37. 37
Coupling Constants
• Distance between the peaks in a multiplet is a
measure of the effectiveness of spin-spin coupling,
and is called as the spin-spin coupling and is called as
the coupling constant(J).
• Measured in Hz.
• Not dependent on strength of the external field.
• Multiplets with the same coupling constants may
come from adjacent groups of protons that split each
other.
38. 38
Carbon-13 NMR
• 12C has no magnetic spin.
• 13C has a magnetic spin, but is only 1% of the carbon in a sample.
• The gyromagnetic ratio of 13C is one-fourth of that of 1H.
• Signals are weak, getting lost in noise.
• Hundreds of spectra are taken, averaged.
• 13C Spectra are easier to analyze than 1H spectra because the
signals are not split. Each type of carbon atom appears as a
single peak.
40. Carbon-13 NMR
• The lack of splitting in a 13C spectrum is a consequence of the low natural
abundance of 13C.
• As splitting occurs when two NMR active nuclei—like two protons are close
to each other. Because of the low natural abundance of 13C nuclei (1.1% of
12C ), the chance of two 13C nuclei being bonded to each other is very small
(0.01%), and so no carbon-carbon splitting is observed.
• A 13C NMR signal can also be split by nearby protons. This 1H-13C splitting is
usually eliminated from the spectrum by using an instrumental technique
so that every peak in a 13C NMR spectrum appears as a singlet.
• The two features of a 13C NMR spectrum that provide the most structural
information are the number of signals observed and the chemical shifts of
those signals.
41. Conti…
• The number of signals in a 13C spectrum gives the number of different
types of carbon atoms in a molecule.
• Because 13C NMR signals are not split, the number of signals equals the
number of lines in the 13C spectrum.
• In contrast to the 1H NMR situation, peak intensity is not proportional to
the number of absorbing carbons, so 13C NMR signals are not integrated.
• In contrast to the small range of chemical shifts in 1H NMR (1-10 ppm
usually), 13C NMR absorptions occur over a much broader range (0-220
ppm).
• The chemical shifts of carbon atoms in 13C NMR depend on the same
effects as the chemical shifts of protons in 1H NMR.
43. Proton decoupling
• Two common broadband decoupling experiments employed in
carbon-13 NMR are broad band decoupling and off resonance
decoupling.
• Broadband decoupling: It is a type of hetronuclear decoupling in
which spin-spin splitting of carbon-13lines by hydrogen-1 is avoided
by irradiating it with a broad-band frequency signal.
• The carbon nuclei see an average of all the possible proton spin
states.
• Thus, each different kind of carbon gives a single, un split peak.
44. Off resonance decoupling
• 13C nuclei are split only by the protons attached directly to them.
• The N + 1 rule applies: a carbon with N number of protons gives a
signal with N + 1 peaks.
45. 45
Interpreting 13C NMR
• The number of different signals indicates the number of
different kinds of carbon.
• The location (chemical shift) indicates the type of functional
group.
• The peak area indicates the numbers of carbons (if
integrated).
• The splitting pattern of off-resonance decoupled spectrum
indicates the number of protons attached to the carbon.
46. Application of NMR spectroscopy
•The important applications of NMR spectroscopy
are following,
•Identification and structural elucidation of
molecules
•Quantitative determination of molecules
47. Application of NMR spectroscopy
1. Structure elucidation: structure of the unknown compound can be
explained from its NMR spectrum.
e.g.
No of main NMR signals should be equal to the no of protons.
Chemical shift indicates that what types of hydrogen bonds are
present.
Spin-spin coupling reveals about the possible arrangments of the
groups.
2. Determination of optical purity:
Since diastereomers differ in their NMR characteristics, so it is used for
practical applications in the determination of optical purity.
48. Application of NMR spectroscopy
3.Keto- Enol Tautomerism: NMR spectroscopy is probably the most
powerful physical analytical method for qualitative and quantitative
analysis of Keto-enol equilibra. E.g. keto and enol forms of acetone.
4.Drug- Macromolecular interaction: study with micele formation by
drugs in solution and drug macromolecular interactions.
• E.g. we can study tetracycline's and metal reaction by NMR.
5.Quantitative analysis: NMR spectroscopy has been used to determine
the molar ratio of the components in a mixture. Hence percentage of
each component can be calculated.
49. Application of NMR spectroscopy
6. Study of isotopes:
Several nuclei in addition to protons which have
magnetic moments can be studied by magnetic resonance technique.
e.g. Fluorine, phosphorus etc.
7.Moisture Analysis:
water absorbed in biological materials such as food
products, appear in NMR spectra as a relatively sharp band.
8.Hydrogen analysis:
percentage of hydrogen in an unknown sample may
be determined easily and rapidly from total integral.
50. Application of NMR spectroscopy
9.Analysis of multicomponent mixtures:
NMR spectroscopy provides a method for the analysis of
multicomponent mixtures.
10.Quantitative organic functional group analysis:
An important application of NMR is the determination of functional
groups, such as hydroxyl groups in alcohol and phenol, and aldehyde
and amides.
11.Elemental analysis: a very important techniques for the elemental
analysis of the compound.
51. General application of NMR
• NMR is used in biology to study the bio fluids, cells, per fused organs
and bio-macromolecules such as nucleic acids (DNA, RNA),
carbohydrates, proteins and peptides.
• NMR is used in physics and physical chemistry to study the high
pressure diffusion, liquid crystals, liquid crystal solutions, membranes
and rigid solids.
• NMR is used in food science.
• NMR is used in pharmaceutical science to study the pharmaceuticals
and drug metabolism.
• NMR is used in chemistry to study the Enantiomeric purity.
52. Application of NMR in medicine
• MRI is an important application of multi-dimentional Fourier
transformation NMR.
• Provides anatomical imaging, measuring the physiological functions,
flow measurements and angiography.
• Tissue perfusion studies and Tumors.
