Nuclear Magnetic Resonance (NMR)

Introduction:

Nuclear Magnetic Resonance (NMR) measures the absorption of electromagnetic

radiation in the radio-frequency region (~4-900 MHz)
- nuclei (instead of outer electrons) are involved in absorption process
- sample needs to be placed in magnetic field to cause different energy
states

NMR was first experimentally observed by Bloch and Purcell in 1946 (received Nobel
Prize in 1952) and quickly became commercially available and widely used.

Probe the Composition, Structure, Dynamics and Function of the Complete Range of
Chemical Entities: from small organic molecules to large molecular weight polymers and
proteins.

NMR is routinely and widely used as the preferred technique to rapidly elucidate the
chemical structure of most organic compounds.

One of the MOST Routinely used Analytical Techniques

 O

Typical Applications of NMR: O NH O

O

1.) Structural (chemical) elucidation O

OH

 Natural product chemistry OH

 Synthetic organic chemistry

HO
O O O
O

- analytical tool of choice of synthetic chemists O

- used in conjunction with MS and IR

2.) Study of dynamic processes Taxol (natural product)
 reaction kinetics
 study of equilibrium (chemical or structural)
3.) Structural (three-dimensional) studies
 Proteins, Protein-ligand complexes
 DNA, RNA, Protein/DNA complexes
 Polysaccharides
4.) Metabolomics
5.) Drug Design
NMR Structure of MMP-13
 Structure Activity Relationships by NMR complexed to a ligand
6.) Medicine -MRI

MRI images of the Human Brain

NMR History
1937 Rabi predicts and observes nuclear magnetic resonance
1946 Bloch, Purcell first nuclear magnetic resonance of bulk sample
1953 Overhauser NOE (nuclear Overhauser effect)
1966 Ernst, Anderson Fourier transform NMR
1975 Jeener, Ernst 2D NMR
1984 Nicholson NMR metabolomics
1985 Wüthrich first solution structure of a small protein (BPTI)
from NOE derived distance restraints
1987 3D NMR + 13C, 15N isotope labeling of recombinant proteins (resolution)
1990 pulsed field gradients (artifact suppression)
1996/7 residual dipolar couplings (RDC) from partial alignment in
liquid crystalline media
TROSY (molecular weight > 100 kDa)
2000s Dynamic nuclear polarisation (DNP) to enhance NMR sensitivity

Nobel prizes
1944 Physics Rabi (Columbia)
1952 Physics Bloch (Stanford), Purcell (Harvard)
1991 Chemistry Ernst (ETH)
2002 Chemistry Wüthrich (ETH)
2003 Medicine Lauterbur (University of Illinois in Urbana ),
Mansfield (University of Nottingham)
NMR History
First NMR Spectra on Water

1H NMR spectra of water

Bloch, F.; Hansen, W. W.; Packard, M. The nuclear induction experiment. Physical Review (1946), 70 474-85.
 NMR History
First Observation of the Chemical Shift

1H NMR spectra ethanol

Modern ethanol spectra

Arnold, J.T., S.S. Dharmatti, and M.E. Packard, J. Chem. Phys., 1951. 19: p. 507.
Bloch Equations

Net Magnetization (M) placed in a magnetic Field (B) will precess:

and relax (R)

and relax individual components:

Course Goals
• We will NOT Cover a Detailed Analysis of NMR Theory
• We will NOT Derive the Bloch Equations
• We will NOT Discuss, in detail, a Quantum Mechanical Description of
NMR
• We will NOT use Product Operator Formulism to Describe NMR
Experiments
• If Interested, Dr. Harbison will Cover These Topics in a Separate
Special Topics Course
• We Will Discuss Practical Aspects of Using an NMR
• We Will Take a Conceptual Approach to Understanding NMR
• A Focus of the Course will be the Application of NMR to Solving the
Structure of Organic Molecules
• We Will Discuss the Basic Operations of an NMR Spectrometer.
• We Will Discuss the Proper Approaches to the Set-up, Process and
Analysis of NMR Experiments
• We Will Use NMR to Solve the Structure of Unknowns
• The Goal is to Not Treat an NMR Spectrometer as a Black
Box, but to be a Competent Experimentalist
Course Goals

To Be Able to Go From This:

To This:
N-(2-Fluoroethyl)-1-((6-methoxy-5-methylpyrimidin-4-yl)methyl)-1H-pyrrolo[3,2-
b]pyridine-3-carboxamide (9) MS (ES+) m/z: 344. 1H NMR (300 MHz, DMSO-d6): δ 2.24 (s,
3H), 3.67 (d, J = 5.46 Hz, 1H), 3.76 (d, J = 5.46 Hz, 1H), 3.93 (s, 3H), 4.50 (t, J = 4.99 Hz, 1H),
4.66 (t, J = 4.99 Hz, 1H), 5.69 (s, 2H), 7.26 (dd, J = 8.29, 4.71 Hz, 1H), 7.94 (d, J = 8.52 Hz,
1H), 8.28 (s, 1H), 8.41 (s, 1H), 8.49 (d, J = 4.57 Hz, 1H), 8.95 (s, J = 5.89, 5.89 Hz, 1H).
HRMS (M + H) calcd for C17H18FN5O2, 344.1517; found, 344.15242.

And Understand How to Read and Interpret the NMR Spectral Data
Course Goals

To Be Able to Go From This:

To This:
Each NMR Observable Nuclei Yields a Peak in the Spectra
“fingerprint” of the structure

2-phenyl-1,3-dioxep-5-ene

1H NMR spectra

13C NMR spectra

 Information in a NMR Spectra

Observable Name Quantitative Information

Peak position Chemical shifts (d) d(ppm) = uobs –uref/uref (Hz) chemical (electronic)
environment of nucleus

Peak Splitting Coupling Constant (J) Hz peak separation neighboring nuclei

(intensity ratios) (torsion angles)

Peak Intensity Integral unitless (ratio) nuclear count (ratio)

relative height of integral curve T1 dependent

Peak Shape Line width Du = 1/pT2 molecular motion

peak half-height chemical exchange
uncertainty principal
uncertainty in energy
A Basic Concept in ElectroMagnetic Theory
Magnets Attract and Align
 A Basic Concept in ElectroMagnetic Theory

A Direct Application to NMR

A moving perpendicular external An electric current in a closed

magnetic field will induce an loop will create a perpendicular
electric current in a closed loop magnetic field
A Basic Concept in ElectroMagnetic Theory

For a single loop of wire, the magnetic field, B

through the center of the loop is:

B
mo I
2R

mo – permeability of free space (4p x 10-7 T · m / A)

R – radius of the wire loop
I – current
A Basic Concept in ElectroMagnetic Theory
Faraday’s Law of Induction
• If the magnetic flux (FB) through an area bounded by
a closed conducting loop changes with time, a current
and an emf are produced in the loop. This process is
called induction.

• The induced emf is:

dF B
 
dt

Simple AC generator
A Basic Concept in ElectroMagnetic Theory

Lenz’s Law
• An induced current has a direction such that the magnetic field of
the current opposes the change in the magnetic flux that produces
the current.

• The induced emf has the same direction as the induced current

Direction of current
follows motion of magnet
Theory of NMR

Quantum Description

Nuclear Spin (think electron spin)

• Nucleus rotates about its axis (spin)
• Nuclei with spin have angular momentum (p) or spin
1) total magnitude

 I ( I  1)
l 1) quantized, spin quantum number I

2) 2I + 1 states: I, I-1, I-2, …, -I

I=1/2: -1/2, 1/2

3) identical energies in absence of external magnetic field

NMR Periodic Table

NMR “active” Nuclear Spin (I) = ½:

1H, 13C, 15N, 19F, 31P

biological and chemical relevance

Odd atomic mass
I = +½ & -½

NMR “inactive” Nuclear Spin (I) = 0:

12C, 16O

Even atomic mass & number

Quadrupole Nuclei Nuclear Spin (I) > ½:

14N, 2H, 10B

Even atomic mass & odd number

I = +1, 0 & -1
 Magnetic Moment (m)
• spinning charged nucleus creates a magnetic field

Magnetic moment

Similar to magnetic field

created by electric current
flowing in a coil

“Right Hand Rule”

• determines the direction of the magnetic field around a current-
carrying wire and vice-versa
Gyromagnetic ratio (g)

• related to the relative sensitive of the NMR signal

• magnetic moment (m) is created along axis of the nuclear spin

2p m m
m g p g 
h I I

where:
p – angular momentum
g – gyromagnetic ratio (different value for each type of nucleus)

• magnetic moment is quantized (m)

m = I, I-1, I-2, …, -I

for common nuclei of interest:

m = +½ & -½
Gyromagnetic ratio (g)

• magnetic moment is quantized (m)

m = I, I-1, I-2, …, -I

for common nuclei of interest:

m = +½ & -½

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
 Magnetic alignment

= g h / 4p

Bo

In the absence of external field, Add a strong external field (Bo) and the nuclear
each nuclei is energetically degenerate magnetic moment: aligns with (low energy)
against (high-energy)
Spins Orientation in a Magnetic Field (Energy Levels)

• Magnetic moment are no longer equivalent

• Magnetic moments are oriented in 2I + 1 directions in magnetic field

 Vector length is:  I ( I  1)

m
 Angle (j) given by: cos j 
I ( I  1)

 Energy given by:

mm
E Bo
I

where,
Bo – magnetic Field
m – magnetic moment For I = 1/2
h – Planck’s constant
Spins Orientation in a Magnetic Field (Energy Levels)

• The energy levels are more complicated for I >1/2

Spins Orientation in a Magnetic Field (Energy Levels)

• Magnetic moments are oriented in one of two directions in magnetic field (for I =1/2)

• Difference in energy between the two states is given by:

DE = g h Bo / 2p

where:
Bo – external magnetic field
h – Planck’s constant
g – gyromagnetic ratio
Spins Orientation in a Magnetic Field (Energy Levels)

• Transition from the low energy to high energy spin state occurs through an
absorption of a photon of radio-frequency (RF) energy

RF

Frequency of absorption: n = g Bo / 2p
NMR Signal (sensitivity)

• The applied magnetic field causes an energy difference between the

aligned (a) and unaligned (b) nuclei

• NMR signal results from the transition of spins from the a to b state

• Strength of the signal depends on the population difference between the a

and b spin states

b
Low energy gap
Bo > 0 DE = h n

a
Bo = 0

• The population (N) difference can be determined from the Boltzmann

distribution and the energy separation between the a and b spin states:

Na / Nb = e DE / kT
NMR Signal (sensitivity)

Since:
DE = hn
and

n = g Bo / 2p
then:

Na / Nb = e DE / kT Na/Nb = e(ghBo/2pkT)

The DE for 1H at 400 MHz (Bo = 9.39 T) is 6 x 10-5 Kcal / mol

Very Small !
Na / Nb  1.000060 ~60 excess spins per
million in lower state
NMR Sensitivity
NMR signal (s) depends on: s % g4Bo2NB1g(u)/T

1) Number of Nuclei (N) (limited to field homogeneity and filling factor)

2) Gyromagnetic ratio (in practice g3)

3) Inversely to temperature (T)

4) External magnetic field (Bo2/3, in practice, homogeneity)

5) B12 exciting field strength (RF pulse)

Na / Nb = e DE / kT DE  g h Bo / 2p

Increase energy gap -> Increase population difference -> Increase NMR signal

DE ≡ Bo ≡ g
NMR Sensitivity

• Relative sensitivity of 1H, 13C, 15N and other nuclei NMR spectra depend on
 Gyromagnetic ratio (g)
 Natural abundance of the isotope
g - Intrinsic property of nucleus can not be changed.

(gH/gC)3 for 13C is 64x (gH/gN)3 for 15N is 1000x

1H is ~ 64x as sensitive as 13C and 1000x as sensitive as 15N !

Consider that the natural abundance of 13C is 1.1% and 15N is 0.37%
relative sensitivity increases to ~6,400x and ~2.7x105x !!

1H NMR spectra of caffeine 13C 13C

NMR spectra of caffeine NMR spectra of caffeine
8 scans ~12 secs 8 scans ~12 secs 10,000 scans ~4.2 hours
NMR Sensitivity

Increase in Magnet
Strength is a Major Means
to Increase Sensitivity
NMR Sensitivity

But at a significant cost!

~$800,000 ~$2,00,000 ~$4,500,000

 NMR Sensitivity

An Increase in concentration is a very

common approach to increase sensitivity

30 mg

1.2 mg

http://web.uvic.ca/~pmarrs/chem363/nmr%20files/363%20nmr%20signal%20to%20noise.pdf
 NMR Sensitivity
But, this can lead to concentration dependent changes in the NMR spectra
(chemical shift & line-shape) resulting from compound property changes

Dimerization as
concentration increases
from 0.4 to 50 mM

Beilstein J. Org. Chem. 2010, 6, No. 3.

H-bond and multimer

formation as concentration
increases from 1 to 30 mM

Beilstein J. Org. Chem. 2010, 6, 960–965.

 NMR Sensitivity

An Increase in the number of scans (NS) or signal-averaging is the most

common approach to increase sensitivity (signal-to-noise (S/N)

S/N ≈ NS1/2
 NMR Sensitivity

But, it takes significantly longer to acquire the spectrum as the number

of scans increase:

Experimental Time = Number of Scans x Acquisition Time

8 scans ~12 secs

10,000 scans ~4.2 hours

 NMR Sensitivity
Lowering the temperature is usually not an effective means of increasing
sensitivity because of chemical shift changes and peak broadening

Significant chemical shift changes

Significant peak broadening

Dalton Trans.,2014, 43, 3601-3611
Theory of NMR

Classical Description

• Spinning particle precesses around an applied magnetic field

A Spinning Gyroscope
in a Gravity Field

m
cos j 
I ( I  1)
Classical Description
• Angular velocity of this motion is given by:

wo = gBo
where the frequency of precession or Larmor frequency is:

n = gBo/2p

Same as quantum mechanical description

Classical Description
• Net Magnetization
 Nuclei either align with or against external magnetic field along the z-axis.
 Since more nuclei align with field, net magnetization (Mo, MZ) exists parallel
to external magnetic field.
 Net Magnetization along +Z, since higher population aligned with Bo.
 Magnetization in X,Y plane (MX,MY) averages to zero.

z z

Mo
x x

y y
Bo Bo
Net Magnetization in a Magnetic Field

z z
Classic View:
- Nuclei either align with or
against external magnetic Mo
field along the z-axis. x x

- Since more nuclei align with y y

field, net magnetization (Mo)
exists parallel to external
Bo Bo
magnetic field

Quantum Description:
- Nuclei either populate low
energy (a, aligned with field)
or high energy (b, aligned b
against field)
Bo > 0 DE = h n
- Net population in a energy
level.
a
- Absorption of radio- Bo
frequency promotes nuclear
spins from a  b.
An NMR Experiment

We have a net magnetization precessing about Bo at a frequency of wo

with a net population difference between aligned and unaligned spins.
z z

Mo
x x

y y
Bo Bo

Now What?

Perturbed the spin population or perform spin gymnastics

Basic principal of NMR experiments
The Basic 1D NMR Experiment

Experimental details will effect the NMR spectra and

the corresponding interpretation
 An NMR Experiment
resonant condition: frequency (w1) of B1 matches Larmor frequency (wo)
energy is absorbed and population of a and b states are perturbed.

z z

Mo B1 off…
x x
B1
(or off-resonance) Mxy
w1
y y
w1

And/Or: Mo now precesses about B1 (similar to Bo)

for as long as the B1 field is applied.

Again, keep in mind that individual spins flipped up or down

(a single quanta), but Mo can have a continuous variation. Right-hand rule
 Classical Description
• Observe NMR Signal
 Need to perturb system from equilibrium.
 B1 field (radio frequency pulse) with gBo/2p frequency
 Net magnetization (Mo) now precesses about Bo and B1
 MX and MY are non-zero
 Mx and MY rotate at Larmor frequency
 System absorbs energy with transitions between aligned and unaligned states
 Precession about B1stops when B1 is turned off

Mz
RF pulse

B1 field perpendicular to B0
Mxy
 Classical Description
• Rotating Frame
 To simplify the vector description, the X,Y axis rotates about the Z axis at
the Larmor frequency (X’,Y’)
 B1 is stationary in the rotating frame

Mz

Mxy
Classical Description
• NMR Pulse
 Applying the B1 field for a specified duration (Pulse length or width)
 Net Magnetization precesses about B1 a defined angle (90o, 180o, etc)

z z
90o pulse
Mo B1 off…
x x
B1
(or off-resonance) Mxy
w1
y y
w1 w1 = gB1
Absorption of RF Energy or NMR RF Pulse

Classic View: z z
- Apply a radio-frequency (RF) 90o pulse
pulse a long the y-axis
Mo B1 off…
x x
- RF pulse viewed as a second
field (B1), that the net B1
(or off-resonance) Mxy
magnetization (Mo) will
w1
precess about with an y y
angular velocity of w1 w1 w1 = gB1
-- precession stops when B1
turned off
b
Quantum Description:
- enough RF energy has been
absorbed, such that the DE = h n
population in a/b are now
equal a
Bo > 0
- No net magnetization along
the z-axis

Please Note: A whole variety of pulse widths are possible, not quantized dealing
with bulk magnetization
Absorption of RF Energy or NMR RF Pulse

A whole variety of pulse 90o

widths are possible, not
quantized dealing with
bulk magnetization
60o

Signal intensity decreases

significantly when not at
optimal 90o pulse length
35o

20o

10o
 An NMR Experiment
What Happens Next?

The B1 field is turned off and Mxy continues to precess about Bo at frequency wo.
z

Mxy wo
y

Receiver coil (x)  NMR signal

FID – Free Induction Decay
Mxy is precessing about z-axis in the x-y plane Time (s)

y y y
Classical Description

• Observe NMR Signal

 Remember: a moving magnetic field perpendicular to a coil will induce a
current in the coil.
 The induced current monitors the nuclear precession in the X,Y plane

Detect signal along X

RF pulse along Y
n = gBo/2p
 NMR Signal Detection - FID
The FID reflects the change in the magnitude of Mxy as
the signal is changing relative to the receiver along the y-axis

Detect signal along X

RF pulse along Y

Again, the signal is precessing about Bo at its Larmor Frequency (wo).

NMR Signal Detection - FID
The appearance of the FID depends on how the frequency
of the signal differs from the Larmor Frequency
Larmor Frequency

Time Domain Frequency Domain

 NMR Signal Detection - Fourier Transform

So, the NMR signal is collected in the Time - domain

But, we prefer the frequency domain.

Fourier Transform is a mathematical procedure that

transforms time domain data into frequency domain
NMR Signal Detection - Fourier Transform
After the NMR Signal is Generated and the B1 Field is Removed, the Net
Magnetization Will Relax Back to Equilibrium Aligned Along the Z-axis

T2 relaxation

Two types of relaxation processes, one in the x,y plane and one along the z-axis

Peak shape also affected by magnetic field homogeneity or shimming

NMR Relaxation
a) No spontaneous reemission of photons to relax down to ground state
Probability too low  cube of the frequency

b) NMR signal relaxes back to ground state through two distinct processes
NMR Relaxation
c) Spin-Lattice or Longitudinal Relaxation (T1)
i. transfer of energy to the lattice or solvent material
ii. coupling of nuclei magnetic field with magnetic fields created
by the ensemble of vibrational and rotational motion of the
lattice or solvent.
iii. results in a minimal temperature increase in sample

Mz = M0(1-exp(-t/T1))

Recycle Delay: General practice is to wait 5xT1 for the system to have fully relaxed.
NMR Relaxation
c) Spin-Lattice or Longitudinal Relaxation (T1)
i. Commonly measure T1 with an inversion recovery pulse sequence

5xT1 fully relaxed

NMR Relaxation

d) spin-spin or transverse relaxation (T2)

i. exchange of energy between excited nucleus and low energy
state nucleus

ii. randomization of spins or magnetic moment in x,y-plane

Mx = My = M0 exp(-t/T2)
NMR Relaxation

d) spin-spin or transverse relaxation (T2)

iii. related to NMR peak line-width

(derived from Heisenberg uncertainty principal)

Please Note: Line shape is also affected by the magnetic fields homogeneity
NMR Relaxation

d) spin-spin or transverse relaxation (T2)

iii. Usually measured with a Carr-Purcell-Meiboom-Gill (CPMG) Pulse sequence

NMR Relaxation

d) Another way to look at T1 and T2

i. T2 is the time it takes a stove to heat a house and T1 is the time it takes for the
heat to be lost to the surrounding environment
ii. If the house is insulated, T1 will be larger than T2
NMR Relaxation
d) One more way to look at T1 and T2
i. Relaxation of 13C occurs through two distinct interactions
ii. Magnetic fields produced by the 1H magnetic dipoles are felt by the 13C spin
• These magnetic fields are modulated by random Brownian motions
(rotational, translational)
iii. Relaxation between 13C and 1H in same molecule - intramolecular relaxation (T2)
iv. Relaxation between 13C and 1H from solvent- intermolecular relaxation (T1)