It is spectroscopy technique to determine number of hydrogen atoms present in the molecules and atoms.It is useful method for separation of molecules and compounds from mixtures components highly recommended in pharmaceutical and chemical engineering fields.

1. Nuclear Magnetic Resonance
(NMR) Spectroscopy
9/3/2021 1
PREPARED BY:-AJAY KUMAR

2. Felix Bloch
1905-1983
Edward M. Purcell
1912-1997
Kurt Wuthrich
1938-
Richard R. Ernst
1933-(Nobel Prize in
1991)
CW NMR 40MHz
1960
9/3/2021
9/3/2021

3. 9/3/2021 3

4. A. Chemical-related research:
1. Analytical tool.
2. Structural characterization of chemical compounds.
B. Material-related research
1. Polymer characterization.
2. C60 (Fullerene).
3. High temperature superconductor research.
4. Heterogeneous catalysis (Ziolite).
5. Surface physics.
C. Study of dynamic processes
1. Reaction kinetics.
2. Study of equilibrium (chemical or structural).
D. Structural (three-dimensional) studies
1. Proteins.
2. DNA/RNA. Protein complexes with DNA/RNA.
3. Polysaccharides
Why bother learning NMR?
9/3/2021 4

5. E. Drug design
1. Structure Activity Relationships (SAR) by NMR
F. Biomedical applications:
1. Metabolic studies of biological systems.
2. Magnetic Resonance Imaging (MRI), diagnostic
imaging, flow imaging, chemical shift imaging, functional
imaging.
3. Macromolecular structure determination in solution.
Finally, it’s the biggest, meanest, most expensive piece of
equipment you’ll see in your career, and this is a great time to
get your hands on it...
Why bother learning NMR?
9/3/2021 5

6. 9/3/2021 6
NMR Historic Review
1924 Pauli proposed the presence of nuclear magnetic moment to explain the
hyperfine structure in atomic spectral lines.
1930 Nuclear magnetic moment was detected using refined Stern-Gerlach
experiment by Estermann.
1939 Rabi et al. First detected unclear magnetic resonance phenomenon by applying
r.f. energy to a beam of hydrogen molecules in the Stern-Gerach set up and
observed measurable deflection of the beam. In 1944 Rabi awarded Nobel
prize in physics
1946 Purcell et al. at Harvard reported nuclear resonance absorption in paraffin
wax.
Bloch et al. at Stanford found nuclear resonance in liquid water.
1949 Chemical shift phenomenon was observed.
1952 Nobel prize in Physics was awarded to Purcell and Bloch, first practical NMR
experiments, which were carried out independently by both of them in 1945 at
different places.
1960 Ernst and Anderson first introduce the Fourier Transform technique into NMR

7. 9/3/2021 7
Late in
1960
Solid State NMR was revived due to the effort of Waugh.
and associates at MIT.
Biological application become possible due to the introduction
superconducting magnets.
NMR imaging was demonstrated.
1970 2D NMR was introduced
1980s Macromolecular structure determination in solution by NMR was
achieved.
1991 Nobel prize in Chemistry was awarded to Richard Ernst for he
developed Fourier transformation method
1990s Continuing development of heteronuclear multi-dimensional NMR
permit the determination of protein structure up to 50 KDa.
MRI become a major radiological tool in medical diagnostic.
2002 Nobel prize in Chemistry was awarded to Kurt Wuthrich for the
elucidation of three-dimensional structures of macromolecules.
2003 Nobel Prizes were awarded to Lauterbach and Mansfield for their
research in magnetic resonance imaging. (MRI)
NMR Historic Review

8. 9/3/2021
Nuclear Spin explanation
• The spinning charged nucleus generates a
magnetic field.
=>
8

9. 9/3/2021
Protons (and other nucleons) have Spin
Spin up (+1/2) Spin down (-1/2)
9

10. 9/3/2021
Each Spinning Proton is Like a “Mini-
Magnet”
Spin up Spin down
N
S
N
S
10

11. The nucleus in free state
9/3/2021 11

12. Nuclear spins
9/3/2021 12

13. 9/3/2021
Principles of NMR
 The nucleus spin on its own axis and magnetic moment is
created, resulting in the precessional orbit with a frequency called
as processional frequency.
 The nucleus of hydrogen atom behaves as spin bar magnet
because it possess both electric and magnetic field.
 NMR involves the interaction between an oscillating magnetic
field of EMR and the magnetic energy of the hydrogen nucleus or
some other nuclei, when they are placed in external magnetic field.
13

14. In any magnetic field, magnetic nuclei like proton precess at a
frequency, v which is proportional to the strength of the applied field.
The exact frequency is expressed by
v=µN Bo/hI
Where Bo =strength of the external field experienced by the proton
I=Spin quantum number
h=Planks constant (6.626x10-34 Js)
µ=Magnetic moment of the particular nucleus
N= Nuclear magnet on constant
L=angular momentum associated with the nuclei
We can think of nuclei as small magnetized tops that spin on their axis:
• The magnetic nuclei has two forces acting on the spins.
•One that tries to turn them towards Bo, and the
other that wants to maintain their angular momentum.
The net result is that the nuclei spins like a top
Bo
wo
m
L
m
L
9/3/2021 14
9/3/2021
Precessional Frequency or Larmor frequency

15. Precession (continued)
•Spins won’t align with Bo, no matter what their intiial orientation was. Spins
pointing ‘up’ and ‘down’ don’t exist!
• Spins will precess at the angle they were when we turned on
the magnetic field Bo:
Bo
There are several magnetic fields acting on the spins. One is Bo, which
is constant in time and generates the precession at wo. The others are
fluctuating due to the molecular anisotropy and its environment, and
make the spins ‘try’ all the possible orientations with respect to Bo in a
certain amount of time.
Orientations in favor of Bo will have lower magnetic energy, and will be
slightly favored.
9/3/2021 15
9/3/2021

16. 9/3/2021
Principles of NMR
 All nuclei in a molecule are surrounded by electron clouds
H effective=H applied - H local
 The local magnetic field can either reinforce the applied
magnetic field, deshield the hydrogen nucleus, and hence a
higher frequency will be required to bring it into resonance.
 The local magnetic field can oppose the applied magnetic field,
shield the hydrogen nucleus from the strength of the magnetic
field and hence a lower frequency will be required to bring it
into resonance.
 Thus slightly different amounts of energy are needed to excite
each individual hydrogen nucleus to its own higher energy
level.
 In summary, different electron densities create different
magnetic environments around each hydrogen atom and
therefore a series of signals are seen across a spectrum.
16

17.  A nucleus with an odd atomic number or an odd mass number has
a nuclear spin.
 The angular momentum of the charge is described as spin number.
They have 0, ½,1, 3/2…etc (I =0 denotes no spin)
 Each proton and neutron has its own spin. If sum of the proton
and neutron is even, spin number (I)= 0,1,2,3, etc
 If sum of the proton and neutron is odd I is half integral=1/2, 3/2,
5/2…etc
Principles of NMR
9/3/2021 17

18.  All nuclei carry a charge and in
some nuclei this charge spins around
an axis generating a magnetic dipole
along the axis of the nucleus.
 1H has a spin I = ½. There are two
allowed spin states –½ and +½. In
the absence of a magnetic field the
two spin states are degenerate and
are equally populated.
 In a magentic field the low energy
spin state is aligned with the
magnetic field and the high energy
opposed to it.
Principles of NMR
9/3/2021 18

19. The case of 1/2 spin nuclei. Presence of external field orients the spins.
Spins that are opposed to the field have higher energy than spins that are
aligned with the field.
Principles of NMR
9/3/2021 19

20. 9/3/2021
NMR equation
• 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.
• Low energy, radio frequency.
• =>
20

21.  The sample absorb different EMR at different frequency.
The spinning axis of the top moves slowly around the
vertical.
 It has been found that the proton precesses about the axis
of the external magnetic field
It has been found that w =  Ho --------(1)
 Where w= angular precessional velocity
Ho =applied field in gauss
 = Gyromagnetic ratio = 2m
hI
9/3/2021 21

22. Here m=magnetic movement of the spinning bar magnet
I = is the spin quantum number of the spinning magnet h=
Planck’s constant
According to the fundamental NMR equation which
correlates
EMR frequencies with the magnetic field we say that
 Ho =2m ------------(2)
Here v is the frequency of EMR
From equation 1) and 2)
Angular precessional velocity w = 2v
9/3/2021 22

23. 1. Precessional frequency –No. of revolution/sec. made by
the magnetic movement vector of the nucleus around the
external field Ho
2. Alternatively it is defined as equal to the frequency of
EMR in megacycles per second necessary to induce
transition from one spin state to another.
3. All nuclei carry a charge , so they will possess spin
angular- momentum.
4. The nuclei which have a finite value of spin quantum
number (I >0) will precess along the axis of rotation.
9/3/2021 23

24. Nuclear spin is the total nuclear angular momentum quantum
number. This is characterized by a quantum number I, which
may be integral, half-integral or 0.
 Only nuclei with spin number I  0 can absorb/emit
electromagnetic radiation. The magnetic quantum number mI
has values of –I, -I+1, …..+I ( e.g. for I=3/2, mI=-3/2, -1/2, 1/2,
3/2 ).
 1. A nucleus with an even mass A and even charge Z 
nuclear spin I is zero. Example: 12C, 16O, 32S  No NMR
signal
2. A nucleus with an even mass A and odd charge Z 
integer value I. Example: 2H, 10B, 14N  NMR
detectable
3. A nucleus with odd mass A  I=n/2, where n is an odd
integer. Example: 1H, 13C, 15N, 31P  NMR detectable
Properties of the Nucleus
9/3/2021 24

25.  To observe resonance, we have to irradiate the molecule with
EMR of the appropriate frequency.
 Different nucleus “type” will give different NMR signal.
Depending on the chemical environment, there are variations on
the magnetic field that the nuclei feels, even for the same type of
nuclei.
The main reason for this is, each nuclei could be surrounded
by different electron environment, which make the nuclei “feel”
different net magnetic field
9/3/2021 25
NMR signals

26.  If the oriented nuclei are now irradiated with EMR of
proper energy, frequency absorption occurs and the lower
energy spin flips to higher energy state.
 When this spin flip occurs, the nuclei are said to be in
Resonance with applied radiation, hence named NMR.
 The exact amount of RF energy necessary for resonance
depends on the strength of external magnetic field and the
nuclei being irradiated.
Strong magnetic field higher energy
Weaker magnetic field less energy
9/3/2021 26

27. 9/3/2021 27

28. 9/3/2021 28
C
C

29. 9/3/2021
Magnetic Shielding
• If all protons absorbed the same amount of
energy in a given magnetic field, not much
information could be obtained.
• But protons are surrounded by electrons that
shield them from the external field.
• Circulating electrons create an induced
magnetic field that opposes the external
magnetic field.
=>
29

30. 9/3/2021
Shielded Protons
Magnetic field strength must be increased for a
shielded proton to flip at the same frequency.
30

31. 9/3/2021
Protons in a Molecule
Depending on their chemical environment,
protons in a molecule are shielded by
different amounts.
=>
31

32. At 60MHZ
1MHZ= 1 million cycles per second to bring 1H nucleus
to resonate
15 MHZ required to bring 13C nucleus to resonate
These energy comparatively less than for which is needed
in IR
For IR 1.1-11K.calories / mol.
NMR 5.7x 10-6K.calories / mol.
Energy used in NMR
9/3/2021 32

33. 9/3/2021
Energy used in NMR
• Em = energy of quantum state m (J)
•  = gyromagnetic ratio (T–1 s–1)
• h = Planck’s constant (6.628  10–34J s)
• B0 = applied magnetic field (T  Tesla)
0
2
B
mh
Em



33

34. 9/3/2021
What factors affect sensitivity?
To enhance sensitivity,
 Increase magnetic field strength, B0
 Choose nucleus with large gyromagnetic ratio, 
 Carry out experiment at low temperature, T
34

35. The chemical shift
The resonant frequency of a certain atom is called chemical
shift.
Advantages:
More compact annotations
Independent on the spectrometer field
In practice, the 1H chemical shifts are in the range 0-10 ppm
or 
The chemical shift depends on:
The atom type (NH, aliphatic CH, aromatic CH, ...)
The amino acid type (Ala, Phe, ...)
The chemical (spatial) environment
9/3/2021 35

36. 9/3/2021
NMR Signals
1. The number of signals shows how many different kinds
of protons are present.
2. The location of the signals shows how shielded or
deshielded the proton is.
3. The intensity of the signal shows the number of
protons of that type.
4. Signal splitting shows the number of protons on
adjacent atoms.
5. To define the position of absorption the NMR chart is
calibrated by using TMS highly shielded molecule. The
exact place on the chart at which a nucleus absorb is
called “chemical shift”
36

37. Chemical shift of TMS is arbitrarily set the zero point
1= 1ppm of the spectrometer operating frequency
1H NMR, 60MHz operating instrument 60,000,000
1= 1ppm (or) 60Hz
100MHZ operating instrument 1= 100Hz
 (ppm)= observed chemical shift (no.of Hz away from TMS)
Spectrometer frequency in MHz
If the induced field, opposes the applied field the electrons
are diamagnetic & the effect is diamagnetic shielding.
9/3/2021 37

38. 9/3/2021
Types of NMR
 Continuous wave (cw) NMR
It detects the resonance of nuclei
 FT NMR
Directly recording the intensity of absorption as a function of
frequency
 The magnet used in NMR produces strong magnetic field
sample probe
1) Sample probe contained between the poles of the magnet
38

39. 9/3/2021
1. The probe has another coil wrapped at right angles to the
transmitter coil
2. Thus the optimum angle for detecting resonance
3. Various temp probe helps to keep the sample at different
temp (-100 to 200°C)
4. So NMR can be used for kinetic study, thermodynamics
39

40. 9/3/2021 40
The chemical shift (δ) is defined as the difference between
the resonance position of a sample nucleus and that of a
standard TMS.
Chemical shift (δ) =[Δν (Hz)/Applied resonance frequency ×
106 Hz)] × 106 ppm
where, Δν = Difference in frequency (Hz) between the
observed signal and that of the standard.
Convention for δ : TMS assigned (δ = 0), values for other
protons are measured positively downfield.
In other words, increasing δ corresponds to increasing de-
shielding of the nucleus.

41. 9/3/2021 41
Spin-spin Interactions
High resolution NMR spectra very often exhibit signals as
multiplets, invariably showing a more or less symmetrical
appearance.
Multiplicity is brought about due to the splitting of the
signal of one set of equivalent nuclei by the magnetic
fields of adjacent sets of nuclei i.e., spin-spin
interactions.

42. 9/3/2021 42
Block Diagram of NMR Spectrometer

43. 9/3/2021
Radiofrequency Oscillator
• It generated by electronic multiplication and natural
frequency of quartz crystal contained in a thermostated
block/different crystal/trasmitters are used
• RFO installed perpendicular to the magnetic field &
transmits RW of fixed frequency 60,100, 200, 300 &
• 500 MHZ
• 1 MHZ is =1 million cycles per second
43

44. 9/3/2021
RF receiver/detector:
a) The sample in the NMR probe gives data which can be
detected as a signal
b) Detectors used in NMR should be sensitive as the
signal levels are small(<1milli volt)
c) So that multiplication of signal is essential
d) Rf receiver also to be perpendicular to the magnet like
oscillator
44

45. 9/3/2021
Recorder
 The signal is sent to the recorder or oscilloscope
 It helps fast scanning of spectrum or electronic filtering of
signal
 The recorder plots resonance signal on y-axis and strength
of magnetic field on x-axis
 The strength of resonance signal  number of nuclei
resonating at that field strength
 Most spectrometer equipped with automatic integrator to
measure the area under the observed signal
45

46. 9/3/2021
Solvents
CDCl3
• Advantages for non-polar to polar compounds
• CDCl3 peak appear in 7.27δ for sparingly soluble
compounds add drop wise (DMSO-d6)
• It will shift residual CHCl3 peak to 8.38δ
CCl4 for non polar compounds any water molecule causes
turbidity
DMSO-d6
• More viscous/restricted rotation, causes line broadening
non-volatile (difficult to remove from the sample)
46

47. 9/3/2021
Additional Factors Affecting Chemical
Shift
 It is very difficult to predict the chemical shift of protons
attached to heteroatoms (O-H, N-H, S-H).
 It is due to hydrogen bonding which has the effect
deshielding the proton and it causes broadening of
signal.
 Replaces the exchangeable protons with deuteriums
which cannot be detected in the proton NMR.
 Hence its chemical shift can be identified without any
problem (It is a useful technique for -OH, NH2 and
COOH
47

48. 9/3/2021
Solvent effect
 Changing the solvent has a dramatic, yet
unpredictable effect on the chemical shift of
signals.
 This is useful if important peaks overlap in a CDCl3
spectrum then another solvent, D6 benzene, D3
acetonitrile etc can be used
48

49. Chemical Shift Data
Different kinds of protons typically come at different chemical shifts.
Shown below is a chart of where some common kinds of protons appear in
the delda scale.
Note that most protons appear between 0 and 10 ppm. The reference,
tetramethylsilane (TMS) appears at 0 ppm, and aldehydes appear near 10
ppm.
ppm
TMS
CH3
CH3
R
O
NR2
CH3
OCH3
R
O
H
R
R R
H
H
R
O
Ph CH3
H
R
Cl
CH3
Ph
OH
OH
R
NH
R
Upfieldregion
of the spectrum
Downfieldregion
of the spectrum
TMS = Me Si
Me
Me
Me
0
1
2
3
4
5
6
7
8
9
10
CH3
HO
(R)
9/3/2021 49

50. 9/3/2021
Location of Signals
• More electronegative
atoms deshield more and
give larger ᵟ values.
• Effect decreases with
distance.
• Additional electronegative
atoms cause increase in
chemical shift.
50

51. 9/3/2021
Typical Values
=>
51

52. 9/3/2021
Aromatic Protons, 7-8
52

53. 9/3/2021
Vinyl Protons, 5-6
=>
53

54. 9/3/2021
Acetylenic Protons, 2.5
=>
54

55. 9/3/2021
Aldehyde Proton,  9-10
=>
Electronegative
oxygen atom
55

56. 9/3/2021
O-H and N-H Signals
Chemical shift depends on concentration.
Hydrogen bonding in concentrated solutions
deshield the protons, so signal is around  3.5
for N-H and  4.5 for O-H.
Proton exchanges between the molecules
broaden the peak.
56

57. 9/3/2021
Carboxylic Acid Proton, 10+
=>
57

58. 9/3/2021
Number of Signals
Equivalent hydrogens have the same chemical shift.
=>
58

59. 9/3/2021
Intensity of Signals
 The area under each peak is proportional to the
number of protons.
 Shown by integral trace.
59

60. HO-CH2-CH3
low
field
high
field
•
Notice that the intensity of peak is proportional to the number of H
9/3/2021 60

61. 1H
13C
Example of 1D : 1H spectra, 13C spectra of Codeine
C18H21NO3, MW= 299.4
9/3/2021 61

62. 9/3/2021
How Many Hydrogens?
When the molecular formula is known, each
integral rise can be assigned to a particular
number of hydrogens.
=>
62

63. The Hard Part - Interpreting Spectra
Following is the NMR spectrum of ethyl acetate.
Since each NMR spectrum is a puzzle
What kinds of data do we get from NMR spectra?
For 1H NMR, there are three kinds each of which we will consider each of
these separately:
1) Chemical shift data - tells us what kinds of protons we have.
2) Integrals - tells us the ratio of each kind of proton in our sample.
3) 1H - 1H coupling - tells us about protons that are near other protons.
9/3/2021 63

64. Integrals
1. Integrals tell us the ratio of each kind of proton.
2. They are lines, the heights of which are proportional to the intensity of
the signal. Example (ethyl acetate) three kinds of protons - CH3 next to
the carbonyl, CH2 next to the O and the CH3 next to the CH2.
3. The ratio of the (height) signals arising from each of these kinds of
protons should be 3 to 2 to 3, respectively.
4. This will help us to identify CH2 signal (it’s the smallest one), but to
distinguish the other two, we have to be able to predict their chemical
shifts. T
5. The CH3 next to the C=O should appear at ~ 2 PPM, while the other
CH3 should be at ~ 1 PPM).
3H'S
3H'S
2 H'S
O
O H H
O CH3
O
H3C O
O
9/3/2021 64

65. 9/3/2021
Spin-Spin Splitting
1. Nonequivalent protons on adjacent carbons have
magnetic fields that may align with or oppose the
external field.
2. This magnetic coupling causes the proton to absorb
slightly downfield when the external field is reinforced
and slightly up field when the external field is opposed.
3. All possibilities exist, so signal is split.
65

66. 9/3/2021
1,1,2-Tribromoethane
Nonequivalent protons on adjacent carbons.
=>
66

67. 9/3/2021
Doublet: 1 Adjacent Proton
=>
67

68. 9/3/2021
Triplet: 2 Adjacent Protons
=>
68

69. 9/3/2021
The N + 1 Rule
If a signal is split by N equivalent protons,
it is split into N + 1 peaks.
=>
69

70. no. of neighbors relative intensities pattern
1
1 1
1 2 1
1 3 3 1
1 4 6 4 1
1 5 10 10 5 1
1 6 15 20 15 6 1
0
1
2
3
4
5
6
singlet (s)
doublet (d)
triplet (t)
quartet (q)
pentet
sextet
septet
example
H
C C
H
H
C C
H
H
H
C C
H
H
H
H
C C
C
H
H
H
H
H
C C
C
H
H
H
H
H
H
C C
C
H
H
H
H
H
H
Splitting Patterns with Multiple Neighboring Protons
If a proton has n neighboring protons that are equivalent, that proton will be
split into n+1 lines. So, if we have four equivalent neighbors, we will have
five lines, six equivalent neighbors… well, you can do the math. The lines
will not be of equal intensity, rather their intensity will be given by Pascal’s
triangle as shown below.
We keep emphasizing that this pattern only holds for when the neighboring
protons are equivalent. Why is that? The answer is two slides away.
9/3/2021 70

71. 9/3/2021
Splitting for Ethyl Groups
=>
71

72. 9/3/2021
Splitting for Isopropyl Groups
=>
72

73. 9/3/2021
Values for Coupling Constants
=>
73

74. 9/3/2021
Complex Splitting
• Signals may be split by adjacent protons,
different from each other, with different
coupling constants.
• Example: Ha of styrene which is split by an
adjacent H trans to it (J = 17 Hz) and an
adjacent H cis to it (J = 11 Hz).
=>
C C
H
H
H
a
b
c
74

75. 9/3/2021
Coupling Constant (J)
 It represents regular multiplets. Actually, J is the
separation (in Hertz ; Hz = sec–1) between the peaks
of regular multiplets.
 The coupling constants help in the identification of the
coupled nuclei because Jac = Jca : and are therefore,
useful in characterizing the relative orientations of
interacting protons.

76. 9/3/2021
Splitting Tree
C C
H
H
H
a
b
c
=>
76

77. 9/3/2021
Spectrum for Styrene
=>
77

78. 9/3/2021
Stereochemical Nonequivalence
 Usually, two protons on the same C are
equivalent and do not split each other.
 If the replacement of each of the protons of a -
CH2 group with an imaginary “Z” gives
stereoisomers, then the protons are non-
equivalent and will split each other.
=>
78

79. 9/3/2021
Some Nonequivalent Protons
C C
H
H
H
a
b
c
OH
H
H
H
a
b
c
d
CH3
H Cl
H H
Cl
a b =>
79

80. 9/3/2021
Hydroxyl Proton
 Ultrapure samples of
ethanol show
splitting.
 Ethanol with a small
amount of acidic or
basic impurities will
not show splitting.
=>
80

81. 9/3/2021
Cl CH2 OH
CH2 3.4- 4.0 and for OH 4.0-5.0 2+1 protons (no splitting)
Acetaldehyde
CH3 2.1 -2.3 and for CHO 9.5-10.1 3+1 protons(no splitting)
N-Pentane
CH3 0.8-1.0 (2+1 protons -triplet) and CH2 1.2-1.4 (multiplet)
Benzene CH 6.5- 8.5 (no splitting)
Toluene
CH3 2.2-2.5 and for CH 6.5-8.5 (no splitting)

82. 9/3/2021
N-H Proton
• Moderate rate of exchange.
• Peak may be broad.
=>
82

83. 9/3/2021
Identifying the O-H or N-H Peak
 Chemical shift will depend on concentration and
solvent.
 To verify that a particular peak is due to O-H or N-H,
shake the sample with D2O
 Deuterium will exchange with the O-H or N-H protons.
 On a second NMR spectrum the peak will be absent,
or much less intense.
83

84. 9/3/2021
Spin-Spin Coupling
 Many 1H NMR spectra exhibit peak splitting
(doublets, triplets, quartets)
 This splitting arises from adjacent hydrogens
(protons) which cause the absorption frequencies
of the observed 1H to jump to different levels
 These energy jumps are quantized and the number
of levels or splittings = n+1 where “n” is the number
of nearby 1H’s
84

85. 9/3/2021
Spin-Spin Coupling
C - Y C - CH C - CH2 C - CH3
H
|
H
|
H
|
H
|
singlet doublet triplet quartet
X Z
X Z X Z X Z
J
85

86. 9/3/2021
Spin Coupling Intensities
1
1 1
1 2 1
1 3 3 1
1 4 6 4 1
1 5 10 10 5 1
1 1
1 1
2
Pascal’s Triangle
1 1
3 3
86

87. 9/3/2021
NMR Peak Intensities
C - CH C - CH2 C - CH3
Y
|
Y
|
Y
|
X Z X Z X Z
AUC = 1 AUC = 2 AUC = 3

88. 9/3/2021
Low Resolution NMR Spectrum of Ethanol,
CH3CH2OH
Chemical shift,  = (r - s) × 106 (ppm)

89. 9/3/2021
High Resolution NMR Spectrum of Ethanol,
CH3CH2OH
Why?

90. 9/3/2021
Modes of NMR Relaxation
• Spin-lattice (longitudinal) relaxation
– characterized by relaxation time T1
• Spin-spin (transverse) relaxation
– characterized by relaxation time T2
• T1 and T2 are typically 0.1-1.0 s

91. 9/3/2021
Spin Lattice Relaxation
• Caused by random fluctuations of nuclei in
sample, whose moving magnetic field can
induce transitions in magnetic moment of
observed nucleus
• T1 is large in solids, much smaller in liquids
• T1 is very short in the presence of
paramagnetic species, or m>½ nuclei

92. 9/3/2021
Spin-Spin Relaxation
• Caused by interaction between nuclei having the
same Larmor frequency, but different energy
states
• Results in dephasing of precessing nuclei,
causing line-broadening of observed nucleus

93. Spin-Lattice Coupling (Nuclear Overhauser Effect)
Two nuclear spins within about 5 Å will
interact with each other through space.
This interaction is called cross-relaxation,
and it gives rise to the nuclear Overhauser
effect (NOE).
Two spins have 4 energy levels, and the
transitions along the edges correspond to
transitions of one or the other spin alone.
W2 and W0 are the cross-relaxation
pathways, which depend on the tumbling
of the molecule.
Nuclear spins can also cross-relax through dipole-dipole interactions and
other mechanisms. This cross relaxation causes changes in one spin through
perturbations of the other spin.
Intensity of the NOE is proportional to r-6 (r is distance between 2 spins).
9/3/2021 93

94. Spin-Lattice Coupling (Nuclear Overhauser Effect)
When two nuclear spins are within 5 Å,
they will cross-relax.
If one spin (S) is saturated (red lines
along the edge), the system is not in
equilibrium anymore.
Magnetization will either flow from the
top to the bottom (W2 active) or from
the right to left (W0 active).
The difference in energy between bb
and aa is twice the spectrometer
frequency, and molecular motions
about that frequency are required for
the transition.
The difference between ab and ba is
very small, and very slow molecular
motions (e.g. proteins) will excite
that transition.
9/3/2021 94

95. 9/3/2021
Different Types of NMR
• Electron Spin Resonance (ESR)
– 1-10 GHz (frequency) used in analyzing free
radicals (unpaired electrons)
• Magnetic Resonance Imaging (MRI)
– 50-300 MHz (frequency) for diagnostic imaging of
soft tissues (water detection)
• NMR Spectroscopy (MRS)
– 300-900 MHz (frequency) primarily used for
compound ID and characterization

96. 9/3/2021
Explaining NMR
UV/Vis spectroscopy
Sample

97. 9/3/2021
Explaining NMR

98. 9/3/2021
A Modern NMR Instrument
Radio Wave
Transceiver

99. Magnet Legs
9/3/2021
NMR Magnet Cross-Section

100. The spectrometer
9/3/2021

101. 9/3/2021

102. 800 MHz
9/3/2021

103. 9/3/2021
NMR Spectroscopy

104. 9/3/2021
The NMR Graph
=>

105. 9/3/2021
NMR Samples

106. 9/3/2021
An NMR Probe

107. 9/3/2021
NMR Sample & Probe Coil

108. 9/3/2021
Types of NMR Tubes
Solid State
Sample Rotors
Solution NMR
Sample Tube
Spinners
NMR Sample
Tubes with Caps

109. 9/3/2021
NMR Sample Preparation
• Use clean + dry NMR tubes and caps
(tubes can be re-used, caps should not!)
• 0.5 ml deuterated solvent
(i.e. CDCl3 ,D2O , Deuterated acetone etc.)
• substrate requirements for routine spectra:
10 mg for proton NMR
100 mg for carbon-13 NMR
• min. filling height of tube: 2 inches (5 cm)
• Cleaning of tubes:
1. rinse with solvent you were using
2. rinse with acetone
3. dry in (vacuum-)oven at low temperature
5 mm

110. 9/3/2021
NMR Sample Preparation
Clean
clear
solution
Suspension
or opaque
solution
Precipitate Not
enough
solvent
Two
phases
Concentration
gradient
GOOD! B a d S a m p l e s !

111. 9/3/2021
GOOD AND BAD NMR SPECTRA
… are the result of:
 Sample preparation
 Choice of solvent
 Homogeneity of magnetic field
 Data acquisition parameters
 Processing procedures

112. 9/3/2021
Good spectrum
ppm
ppm

113. 9/3/2021
Bad spectrum ?

114. 9/3/2021
Bad spectrum !
Tall signals
are cut off

115. 9/3/2021
Bad spectrum ?

116. 9/3/2021
Bad spectrum !
Signals too small
(only allowed to compare signal
intensities between different spectra)

117. 9/3/2021
Bad spectrum ?

118. 9/3/2021
Bad spectrum !
Broad signals
(bad sample, poor shimming,
wrong processing parameters)

119. 9/3/2021
Bad spectrum ?

120. 9/3/2021
Bad spectrum !
Signals are distorted
(automatic phase correction
is often insufficient)
Excessive peak picking
(low p.p. threshold,
also due to improper phasing)

121. 9/3/2021
Applications
• Determination of exact structure of drugs
and drug metabolites - MOST POWERFUL
METHOD KNOWN
• Detection/quantitation of impurities
• Analysis/deconvolution of liquid mixtures

122. 9/3/2021
 Analysis of blood, urine and other biofluid
mixtures to quantify and identify metabolite
changes
 Allows one to detect drug toxicity and even
localize toxicity (for preclinical trials) in a non-
invasive way
 Detection, identification and quantitation of
primary and secondary drug metabolites

123. 9/3/2021
Other Applications
• Clinical testing (detection of inborn errors of
metabolism, cancer, diabetes, organic solvent
poisoning, drugs of abuse, etc. etc.)
• Cholesterol and lipoprotein testing
• Chemical Shift Imaging (MRI + MRS)
• Pharmaceutical Biotechnology (proteins,
protein drugs, SAR by NMR)

124. 9/3/2021
Hydrogen and Carbon Chemical Shifts
=>

125. Acknowledgement
For lecture notes refer
•http://redpoll.pharmacy.ualberta.ca
•http://www.pharmacy.ualberta.ca/pharm325
9/3/2021