NMR Spectroscopy
Nuclear magnetic resonance spectroscopy:
commonly referred to as NMR, is a technique
which exploits the magnetic properties of
certain nuclei to study physical, chemical, and
biological properties of matter
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)
Continuation of NMR History
Nobel prizes
1944 Physics Rabi (Columbia)
1952 Physics Bloch (Stanford),
Purcell (Harvard)
"for his resonance
method for recording
the magnetic
properties of atomic
nuclei"
1991 Chemistry Ernst (ETH)
"for his contributions to
the development of the
methodology of high
resolution nuclear
magnetic resonance
(NMR) spectroscopy"
"for their development
of new methods for
nuclear magnetic
precision measurements
and discoveries in
connection therewith"
Continuation of NMR History
2002 Chemistry Wüthrich (ETH)
"for his development of nuclear magnetic
resonance spectroscopy for determining the
three-dimensional structure of biological
macromolecules in solution"
2003 Medicine Lauterbur (University of Illinois in
Urbana ), Mansfield (University of Nottingham)
"for their discoveries concerning
magnetic resonance imaging"
Basic Concepts of NMR
NMR stems from the same basic principle as all other forms
of spectroscopy. Energy interacts with a molecule, and
absorptions occur only when the incident energy matches the
energy difference between two states
NMR basically a non-destructive analytical technique
which is based on magnetic properties of nuclei of interest
1H
NMR (Proton NMR): Gives information about types of
protons and their immediate environment
13C
NMR (Carbon NMR): Gives direct information about
the carbon skeleton of an organic molecule
The Nuclear Magnet
A spinning charge, whether positive or negative, constitutes a
circular electric current which generates a magnetic dipole along
the spin axis.
Since all nuclei carry charge, a spinning nucleus behaves as a tiny
bar magnet, called Nuclear Magnet, placed along the spin axis,
and has a characteristic magnetic moment, µ.
Nuclear Spin
An intrinsic form of angular momentum possessed by atomic
nuclei containing an odd number of nucleons (protons or
neutrons).
The Nuclear Spin is different from the electron spin. The nuclear
spin represents the total angular momentum of the nucleus. It is
represented by symbol, I. The nucleus is, although, composed of
neutrons and protons but it acts as if it is a single entity which has
intrinsic angular momentum.
The nuclear spin depends on the mass number, if the mass
number is odd then the nucleus has half-integer spin like the
electron while if the nucleus has even mass number then its spin
will be integer spin.
Nuclear Spin Quantum Number (I)
Quantum Numbers are a set of values which describes the state
of any electron including the orientation and type of orbital
where it is likely to be found, its spin and its distance from the
nucleus.
The nuclear spin quantum number I, determines the allowed spin
states of the nucleus and it is represented by;
Allowed Nuclear Spin States = 2 I + 1
In the case of hydrogen, l = 12 and hence the allowed spin states of
the nucleus is 2. Similarly if the value of l of an element is known
we can find the allowed states of the nucleus of that atom.
Nuclear Spin States
The nuclear spin states can take any number, fraction or integer.
The number is dependent on the three points,
1. If both the neutrons and the protons in the nucleus are even
in number then the nucleus has NO spin states.
2. If the sum of the neutrons and protons in the nucleus is odd
then the nucleus has half integer spin (1/2. 3/2, 5/2, …)
3. If both the neutrons and the protons in the nucleus are odd in
number then the nucleus has an integer spin states (1, 2, 3, …)
In other words the nucleus with odd number of protons or
neutron or both should have the nuclear spin while if both are
even then there is no nuclear spin.
Spin of Nuclei
Fermions : Odd mass nuclei with an odd number of nucleons have
fractional spins.
I = 1/2 ( 1H, 13C, 19F, 31P ), I = 3/2 ( 11B, 33S ) & I = 5/2 ( 17O ).
Bosons : Even mass nuclei with odd numbers of protons and
neutrons have integral spins.
I = 1 ( 2H, 14N )
Even mass nuclei composed of even numbers of protons
and neutrons have zero spin
I = 0 (12C, and 16O, 32S)
Nuclear Spin Resonance / Spin Flipping
The Nuclear Spin Resonance is study of the magnetic nuclei under
the influence of the external magnetic field. The NSR is the
phenomena used in many scientific instruments and experiment, in
which a magnetic nucleus absorbs and emits the electromagnetic
radiations under the influence of the external magnetic field.
In the normal condition the nuclear spin is randomly oriented but
under the influence of the external magnetic field the nuclear
spin either aligns in the same direction with the applied magnetic
field or aligns in the opposite direction with the applied magnetic
field. The nuclear spin aligned with the magnetic field has lower
energy than the nuclear spin aligned opposite to the magnetic
field.
Energy Differentiation
In the presence of an external magnetic field (B0), two spin states
exist, +1/2 and -1/2 (For I=1/2).
The magnetic moment of the lower energy +1/2 state (α)is aligned
with the external field, and that of the higher energy -1/2 ( β)spin state
is opposed to the external field.
The energy difference (ΔE) between these spin states is directly
proportional to the strength of the applied magnetic field, Ho, and is
given as:
ΔE = hγHo / 2π
as ΔE = hν
In terms of frequency
ν = γHo / 2π
Where γ is proportionality constant which determines sensitivity of a
nucleus to the applied magnetic field and is a measure of magnetic
strength of the nuclear magnet.
Magnetogyric ratio (γ)
In fact, γ is the ratio between the magnetic moment (µ) and spin
quantum number (I) of a nucleus, called magnetogyric ratio and is
characteristic of each nucleus.
γ = 2πµ / h I
For a proton, γ has the value of 26, 764 radians / gauss . Sec
Even in a very strong magnetic field, the energy difference between
the two spin states of proton is very small. For example, in a magnetic
field of 23, 500 gauss (2.35 T), the energy difference (ΔE) between
the spin states of proton is:
ΔE = hγHo / 2π
= 6.625 x 10-34 J.s x 26764 G-1.s-1 x 23500 G / 2 x 3.143
= 6.625 x 10-26 J
This is a small amount of energy and corresponds to the radiowave
region of the electromagnetic spectrum.
Mechanism of Energy Absorption
during Spin Flipping
Larmor Precession / Wobbling
In addition to alignment of nuclei with a magnetic moment, application of an
external magnetic field will produce a secondary spin or wobble (precession) of
nuclei around the main or static magnetic field. The precessional path around the
magnetic field is circular like a spinning top.
Larmor or Precessional Frequency
Spinning particle precesses about the
external field axis with an angular
frequency known as the Larmor frequency
wL = g Bo
When radio frequency energy matching
the Larmor frequency is introduced at a
right angle to the external field, it would
cause a transition between the two energy
levels of the spin. In other world, the
precessing nucleus will absorb energy and
the magnetic moment will flip to its I =
_1/2 state
Precessional Frequency for Some Nuclei at
1T
Isotope
Net Spin
g / MHz T-1
Abundance / %
1H
1/2
42.58
99.98
2H
1
6.54
0.015
3H
1/2
45.41
0.0
31P
1/2
17.25
100.0
23Na
3/2
11.27
100.0
14N
1
3.08
99.63
15N
1/2
4.31
0.37
13C
1/2
10.71
1.108
19F
1/2
40.08
100.0
Precessional Frequency of Proton at
Various Magnetic Field Strengths
Precessional Frequency of 13C
The precessional frequency of a nucleus depends upon its
Magnetogyric ratio (γ) and strength of applied magnetic field (Bo).
wL = g Bo
Therefore, in a magnetic field of 23, 500 gauss (2.35 T) a 1H having
magnetogyric ratio (26, 764 radians G-1 S-1) precesses with a frequency
of 100 MHz.
Whereas the precessional frequency of 13C in the same magnetic field
will be 25.2 MHz because the magnetogyric ratio for 13C (6, 728
radians G-1 s-1) is about one-fourth of that of 1H.
Relationship between Frequency and
Magnetic Field Strength
Population Distribution
In a given sample of a specific NMR-active nucleus, the nuclei will be
distributed throughout the various spin states available. As the energy
separation between these states is comparatively small, energy from thermal
collisions is sufficient to place many nuclei into higher energy spin states.
The number of nuclei in each spin state is described by
the Boltzmann distribution :
Nupper /Nlower = e-γBo/kT
For example, at room temperature in a given sample of 1H nuclei in an
external magnetic field of 1.41 Tesla the ratio between upper energy
and lower energy population is only 0.9999382.
Population Distribution
Effect of a radio frequency
(Spin Relaxation)
Radiationless Spin Relaxation
A nucleus in the higher energy spin state returns to the lower energy spin state,
by losing the excess energy to some magnetic vector present in the surrounding.
Spin-Lattice relaxation (T1) / Longitudinal relaxation
Lattice refers to the framework of molecules which may belong to sample or
solvent in any form
All these molecules have nuclei precessing therefore a variety of magnetic fields
are present in lattice
A small magnetic field properly oriented in the lattice and precessing with a
comparable frequency can induce transition
‹ pin-lattice relaxation process causes an excess of nuclei in the lower energy
S
spin state
Spin-lattice relaxation depends on the nature and the environment of the nucleus
being observed and is useful in the structure elucidation
Radiationless Spin Relaxation
Spin-Spin relaxation (T2) / Transverse Relaxation
Spin-spin relaxation occurs by the mutual exchange of energy by two nuclei
precessing in the different spin states but the same frequency, in close
proximity of each other.
It continues even in the absence of radio-frequency, individual nuclei convert
from once spin state to another quite readily because they experience the
magnetic field of other nearby nuclei.
Although it shortens the life-time of individual nuclei in the higher energy spin
state, it does not contribute to the restoration of low energy state population
excess
Spin Relaxation
Instrumentation of NMR
Two types of instruments are used in NMR studies
Continuous Wave (CW)
The spectrometer scans through a range of frequencies
‹The sample is interrogated with one frequency at a time
‹As the frequency range is scanned, a plot of signal intensity vs. frequency is
generated
Fourier Transform (FT)
The sample is interrogated with a range of frequencies “all at once”
The decay of the signal over time is observed as a
Free Induction Decay (FID) Plot
‹A Fourier transform changes the signal vs. time plot
into a signal vs. frequency plot.
Continuous Wave NMR
Field Sweep Mode: a constant frequency, which is continuously on, probes the
energy levels while the magnetic field is varied. The energy of this frequency is
represented by the blue line in the energy level diagram.
Frequency Sweep Mode: The CW experiment can also be performed with a
constant magnetic field and a frequency which is varied. The magnitude of the
constant magnetic field is represented by the position of the vertical blue line in
the energy level diagram.
Fourier Transform NMR
In FT-NMR, all frequencies in a spectrum are irradiated simultaneously
with a radio frequency pulse. Following the pulse, the nuclei return to
thermal equilibrium. A time domain emission signal is recorded by the
instrument as the nuclei relax. A frequency domain spectrum is
obtained by Fourier transformation.
Advantages of FT over CW
I‹f a signal is weak, many scans must be averaged to enhance the
signal/noise ratio.
Signal/noise ratio rises as the square root square root of number of scans
Only FT instruments can obtain a large number of scans in a reasonable
time.
‹
A scan can be performed much more quickly.
CW-NMR: 5 minutes; FT-NMR: 5 seconds.
Components of an NMR
Spectrophotometer
Powerful Magnet
Field Sweep Generator
Sample Tube
Radio-frequency Transmitter
Radio-frequency Receiver
Amplifier
Recorder
Integrator
Instrumentation of NMR
Schematic NMR Spectrometer
Instrumentation of NMR
NMR Spectrophotometers are available in wide-ranging magnetic field
strengths.
Often described in terms of the resonance frequency of proton
Do all the protons resonate at same
frequency?
Shielding of Proton
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.
Shielded Protons
Magnetic field strength must be increased for
a shielded proton to flip at the same
frequency.
Protons in a Molecule
Depending on their chemical environment,
protons in a molecule are shielded by
different amounts.
=>
NMR Output
Peaks appear at the positions of absorption, also called the positions of resonance, for different
nuclei in the molecule. The exact chemical shift of a particular nucleus in a molecule gives us
information about how the atom with that nucleus is bonded in the molecule. The x-axis of the
spectrum is called the delta scale (δ) with units of ppm and the y-axis is an intensity scale. The
area under the peak on the y-axis is proportional to the number of 1H nuclei in the molecule with
the same chemical shift.
Reference Standard
TMS is added to the sample.
Since silicon is less electronegative than
carbon, TMS protons are highly shielded.
Signal defined as zero.
Organic protons absorb downfield (to the
left) of the TMS signal.
Chemically inert
Miscible with most of the organic liquids
Low boiling point (27o C)
All protons are magnetically equivalent
For aqueous samples
Sodium 2,2-Dimethyl- 2-Silapentane5-Sulfonate (DSS)
The Chemical Shift
NMR absorptions generally appear as sharp peaks.
Increasing chemical shift is plotted from right to left.
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.
NMR absorptions are measured relative to the position of a reference peak at 0
ppm on the delta scale due to tetramethylsilane (TMS). TMS is a volatile inert
compound that gives a single peak upfield from typical NMR absorptions
The frequency of absorption for a nucleus of interest relative to the frequency of absorption
of a molecular standard is called the chemical shift of the nucleus.
NMR Signals
• The number of signals shows how many
different kinds of protons are present.
• The location of the signals shows how
shielded or deshielded the proton is.
• The intensity of the signal shows the number
of protons of that type.
• Signal splitting shows the number of protons
on adjacent atoms.
Number of Signals
The number of NMR signals equals the number of different types of protons in
a compound.
Protons in different environments give different NMR signals.
Equivalent protons give the same NMR signal.
Sample Handling
Complete homogeneity of the magnetic field
Small sample ~ 1 mL of 5% sample solution in a suitable solvent
3-4 cm depth in a glass tube about 15 cm long and 5 mm wide
Few percent of reference standard is also added to sample solution
Liquid sample directly should not be viscous
Chemical shift values may have difference in different solvents (~ 1 ppm)
Solvents should be mentioned
Non-viscous, inexpensive, capable of dissolving sample, chemically inert,
devoid of protons CCl4, CS2, C6D6, CD3CN, (CD)2SO
Deuterated solvents may give a small additional peak
Sample Handling
The δ Value
The δ Value
Relative Peak
Area
Molecular Structure and Chemical Shifts
•Protons in different environments experience
different degrees of shielding and thus have
different chemical shift values
•Shielding of a proton is determined by the
structure of the molecule an is influenced by two
types of intra-molecular effects
1. Local diamagnetic effect / Effect of
electronegativity
2. Magnetic anisotropic effect
•In fact the shielding that a proton experiences is
a combination of these two types of shielding
effects
Local Diamagnetic/Electronegativity Effect
•Local diamagnetic shielding is due to the circulation of electrons around the proton
itself.
•It therefore depends upon electron density in the immediate vicinity of the proton
which in tern depends upon the electronegativity of the atom to which the proton is
attached.
Methyl Halides-Within a group
Methyl Halides-Within a period
Methyl Halides- Cumulative effect
Magnetic Anisotropic Effect
The chemical shift values for ethane, ethylene and acetylene would increase in
the same order in a regular way…
Acetylene
sp
>
Ethylene
sp2
>
Ethane
sp3
But in actual…
the directional property of the induced magnetic field of π-electrons
Therefore a proton may feel additional shielding or de-shielding depending on its orientation
relative to the induced magnetic field caused by the circulation of electrons originating from
the other parts of the molecule.
Such effects are called magnetic anisotropic effects.
The local diamagnetic effects operate only along a chain of the atoms, the magnetic
anisotropic effects operate through space no matter whether the influenced group is
directly attached to the anisotropic group or not.
Why acetylene has a lower chemical shift value from that of ethylene?
The same phenomenon can also de-shield a proton.
4-ethynylphenanthrene; H-5 proton is 1.7 ppm
downfield from…
Similar arguments can be put forward to explain the unexpectedly high chemical shift for
the ethylenic protons and for the aldeydic protons.
The Ring Current Effect: Protons of the benzene ring are highly de-shielded…
Due to ring current effect the protons or any groups in the plane of the ring will be highly
de-shielded while immediate above and below the ring will be highly shielded.
An interesting example of the manifestation of the ring-current effect…
The six protons inside the ring are strongly shielded (δ = -2.88 ppm) downfield from TMS
While the twelve protons outside the ring are highly de-shielded (δ = 9.25 ppm)
The ring current effect in aromatic compounds (Cyclically delocalized π- electrons) has
greater deshielding effect as compared to the conjugated alkene groups. And this criterion
can be used to determine the aromatic character of an organic molecule.
δ = 2.34 ppm
δ = 1.95 ppm
ortho-Hs
δ = 7.85 ppm
meta and para-Hs
δ = 7.40 ppm
Acetophenone
Effect of H-bonding
H-bonding causes transfer of the electron-cloud from the hydrogen atom to a
neighboring electronegative atom (O, N or S), resulting in deshielding of the
hydrogen atom.
The greater the extent of the H-bonding, the greater the deshielding of the
proton and its signal will be downfield i.e. a higher chemical shift.
Depends upon the type of solvent used, dilution and temperature.
Neat Ethanol
5.53 ppm
(20%) in CCl4
2-4 ppm
Very dilute/Vapour
0.5 ppm
Since, Intra-molecular H-bonding is not affected by dilution, temperature
and solvent changes their chemical shift virtually remains constant.
Deuterium Exchange
Proton Chemical Shift Ranges
•Electronegative groups are "deshielding" and tend to move NMR signals from neighboring protons further "downfield" (to higher ppm values).
•Protons on oxygen or nitrogen have highly variable chemical shifts, which are sensitive to concentration, solvent, temperature, etc.
•The -system of alkenes, aromatic compounds and carbonyls strongly deshield attached protons and move them "downfield" to
higher ppm values.
Spin-Spin Coupling
Spin relaxation