The document discusses nuclear magnetic resonance (NMR) spectroscopy. It provides information on: 1) NMR spectroscopy gives structural information about molecules, including the number and types of atoms. It can determine 3D structures of proteins and nucleic acids. 2) Applications include studying protein dynamics, hydrogen bonding, and protein-ligand interactions. Magnetic resonance imaging uses NMR principles to detect tumors. 3) NMR signals provide information on the chemical environment and number of equivalent protons in a molecule.

1. Nuclear magnetic Resonance
(NMR) Spectroscopy
Compiled by
Dr. Heena Dave
Institute of Science
Nirma University

2. • Structure of small flexible molecules that cannot be crystallized (peptides,
oligosaccharides, ...)
• 3D structure determination of proteins, nucleic acids, protein/DNA
complexes, ...)
• dynamics (ps to s)
• electrostatics (pKa values)
• hydrogen bonding (NH temperature coefficients, H2O/D2O exchange)
• unfolded/partially folded states of proteins
• bound solvent
• protein/ligand interactions (also very weak)
• diffusion coefficients
• analysis of biomolecules in vivo
Applications of NMR

3. Protein structure calculation by NMR
Cloning → expression (labeling) → purification

sequential assignments  data acquisition

side-chain assignments → NOE assignments

list of geometrical restraints

structure calculation → structure refinement

structure/function relationships  validation
(electrostatic potentials,
surface analysis, ligand binding sites, ...)

4. Magnetic resonance imaging
• Noninvasive
• Type of NMR spectroscopy.
• Only protons in one plane can be in
resonance at one time.
• Computer puts together “slices” to get
3D.
• Tumors are readily detected by this
method.

6. It is a spectroscopic technique that gives us
information about the number and types of atoms in a
molecule:
• hydrogen using 1H-NMR spectroscopy
• carbon using 13C-NMR spectroscopy
• phosphorus using 31P-NMR spectroscopy
• silicon using 29Si-NMR spectroscopy
• 19F, 119Sn, 195Pt, ...
NMR

8. Nuclear Spin States
• The allowed nuclear spin states are determined by the spin quantum number, I, of
the nucleus.
• A nucleus with spin quantum number I has 2I + 1spin states. If I = 1/2, there are two
allowed spin states.
Spin quantum numbers and allowed nuclear spin states for selected isotopes of
elements common to organic compounds:

9. Nuclear spins in zero
magnetic field:
• Normally, nuclear spins are
completely random in orientation.
• The number of allowed nuclear spin
states does not change for the
element.
• When placed in an external
magnetic field of strength B0, only
certain orientations of nuclear
magnetic moments are allowed.
• They can line up with or against
the field by spinning clockwise or
counter clockwise.
Nuclear spins in a
magnetic field:

10. • Alignment with the magnetic field (called ) is lower energy than
against the magnetic field (called ). The energy difference depends
on the strength of the magnetic field
• For nuclei that don’t have spin, such as 12C, there is no difference in
energy between alignments in a magnetic field since they are not
magnets. Therefore, we can’t do NMR spectroscopy on 12C.

12. • When nuclei with a spin quantum number of 1/2 are placed in an
applied field, majority of nuclear spins are aligned with the applied
field in the lower energy state.
• This equilibrium alignment can be changed to an excited state by
applying radio frequency pulses.
• If a nucleus is irradiated with electromagnetic radiation of the
appropriate energy,
- the energy is absorbed, and
- the nuclear spin is flipped from spin state +1/2 (with the
applied field) to -1/2 (against the applied field).
Nuclear Magnetic Resonance

13. Nuclear Magnetic Resonance
• Resonance: the absorption of electromagnetic radiation by a nucleus and the flip of its
nuclear spin from a lower energy state to a higher energy state.
• The instrument detects this and records it as a signal.

14. • In an applied field strength of 1.41T,
- 1H is approximately 0.00572 cal/mol, which corresponds to electromagnetic radiation of 60 MHz
(60,000,000 Hz).
- 13C is approximately 0.00143 cal/mol, which corresponds to electromagnetic radiation of 15 MHz
(15,000,000 Hz).
• In an applied field strength of 7.05T,
- 1H is approximately 0.0286 cal/mol, which corresponds to electromagnetic radiation of 300 MHz
(300,000,000 Hz).
- 13C is approximately 0.00715 cal/mol, which corresponds to electromagnetic radiation of 75 MHz
(75,000,000 Hz).

15. E
Bo
E = h x 300 MHz E = h x 500 MHz
7.05 T 11.75 T
 proton spin state
(lower energy)
 proton spin state
(higher energy)
Graphical relationship between
magnetic field (B o) and frequency ( )
for 1
H NMR absorptions
at no magnetic field,
there is no difference beteen
- and - states.
0 T
In a magnet of 7.05 Tesla, it takes EM radiation of about 300 MHz (radio waves).
So, if we bombard a molecule with 300 MHz radio waves, the protons will absorb that
energy and we can measure that absorbance.
In a magnet of 11.75 Tesla, it takes EM radiation of about 500 MHz (stronger magnet
means greater energy difference between the - and - state of the protons)

19. NMR Signals
• The number of signals show how many different kinds of protons are present.
• The location of signals show shielded or deshielded the proton is.
• The intensity of signal shows the number of protons of that type.
• Signal splitting shows the number of protons on adjacent atoms.
• The circulation of electrons around a nucleus in an applied field is called
diamagnetic current and the nuclear shielding resulting from it is called
diamagnetic shielding.
• The difference in resonance frequencies among the various hydrogen or
carbon
nuclei within a molecule due to shielding or deshielding is generally very
small.

20. • It is customary to measure the resonance frequency (signal) of individual nuclei
relative to the resonance frequency (signal) of a reference compound.
• The reference compound now universally accepted is tetramethylsilane (TMS).

22. The Chemical Shift () scale
• We take the standard compound (TMS) to standardize NMR
instruments.
• An NMR of that standard is taken and its absorbance frequency is
measured.
• We then measure the frequency of our sample and subtract its
frequency from that of the standard.
• This is then divided by the frequency of the standard.
• This gives a number called the chemical shift, (), which does
not depend on the magnetic field strength.

23. If we have a magnet where a standard absorbs at 300,000,000 Hz (300 megahertz), and the
sample absorbs at 300,000,300 Hz. The difference is 300 Hz, so we take 300/300,000,000 =
1/1,000,000 and call that 1 part per million (or 1 PPM).
If the same sample is placed in a stronger magnetic field where the reference comes at
500,000,000 Hz, or 500 megahertz. The frequency of the sample will increase proportionally,
and will come at 500,000,500 Hz. The difference is now 500 Hz, but we divide by
500,000,000 (500/500,000,000 = 1/1,000,000, = 1 PPM).
So there is no difference.
The Chemical Shift () scale
Chemical shift (): the shift of an NMR signal from the signal of TMS. Normally
given in parts per million (ppm).
Downfield: the shift of an NMR signal to the left on the chart paper.
Upfield: the shift of an NMR signal to the right on the chart paper.

25. Equivalent Atoms
Equivalent atoms have the same chemical environment and may be related by
symmetry. Equivalent hydrogens will have the same chemical shift.
Molecules with:
• 1 set of equivalent atoms give 1 NMR signal.
• 2 or more sets of equivalent atoms give a different NMR signal for each set.

26. Chemical shift depends upon:
• Electron density – induced magnetic field – shielding and deshielding.
Electron density depends upon:
- electronegativity of nearby atoms - More electronegative atoms deshield more and give
larger shift values.
- effect decreases with distance
- Additional electronegative atoms cause increase in chemical shift.
• Hybridization of nearby atoms
• magnetic induction within an adjacent  bond
Electronegativity Effects

28. Hibridization Effects

29. Adjacent  Bonds Effects

30. Pi electrons of carbon-carbon triple bond
shields an acetylenic hydrogen and shifts its
signal upfield (to the right) to a smaller  value.

31. Pi electrons of a carbon-carbon double bond
deshields vinylic hydrogens and shifts their
signal downfield (to the left) to a larger  value.

32. Pi electrons in an aromatic ring deshields
aromatic hydrogens and shifts their signal
downfield (to the left) to a yet larger  value.
This extra inductive effect is known as the ring
current and is seen in a wide variety of aromatic
rings.

35. Proton-Proton Coupling, J-Coupling

36. C C
HB
HA
HA HB
HA is split into two lines because
it feels the magnetic field of HB.
HB is split into two lines because
it feels the magnetic field of HA.
For this line, HB is lined up
with the magnetic field
(adds to the overall
magnetic field, so the line
comes at higher frequency)
For this line, HB is lined up
against the magnetic field
(subtracts from the overall
magnetic field, so the line
comes at lower frequency)
C C
HB
HA
HA'
HA + HA' HB
HA and HA' appear at the same
chemical shift because they are
in identical environments
They are also split into two lines
(called a doublet) because they
feel the magnetic field of HB.
HB is split into three lines
because it feels the magnetic
field of HA and HA'
Note that the signal produced
by HA + HA' is twice the size
of that produced by H
B

37. HB
Now, let's "turn on"HB - HA coupling. This splits
the single line into two lines
If uncoupled, H
B would appear as a
singlet where the dashed line indicates
the chemical shift of the singlet.
Now, let's "turn on"HB - HA' coupling. This
splits each of the two new lines into two lines,
but notice how the two lines in the middle
overlap. Overall, we then have three lines.
C C
HB
HA
HA'
Why three lines for HB ?

38. 1,1,2-Tribromoethane
Nonequivalent protons on adjacent carbons.

39. Doublet: 1 Adjacent Proton

40. Triplet: 2 Adjacent Protons

41. The N + 1 Rule
If a signal is split by N equivalent protons,
it is split into N + 1 peaks.

44. •Protons on carbon-carbon double bonds
often give characteristic splitting
patterns.
•A disubstituted double bond can have
two geminal protons, two cis protons, or
two trans protons.
•When these protons are different, each
proton splits the NMR signal of the
other so that each proton appears as a
doublet.
•The magnitude of the coupling constant
J for these doublets depends on the
arrangement of hydrogen atoms.

47. For the spectrum of vinyl acetate. Since Hc and Hb are not
equivalent to each other, we cannot just add them
together and use the n + 1 rule.
When two sets of adjacent protons are different from
each other and couple to a common set of protons with
different J, (n protons on one adjacent carbon and m
protons on the other), the number of peaks in an NMR
signal = (n + 1)(m + 1).

48. Interactions between magnetic nuclei
Two Types:
Through space and through bonds

49. The 2D spectrum
• 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.
f1
f2

50. NMR Spectrometer

51. • Components: a powerful magnet, a radio-
frequency generator, and a radio-
frequency detector.
• The sample is dissolved in a solvent, most
commonly CDCl3 or D2O, and placed in a
sample tube which is then suspended in the
magnetic field and set spinning.
• Deuterated solvents are used to eliminate
1H signals from the solvent.
NMR Spectrometer