Instrumental Analysis

NUCLEAR MAGNETIC RESONANCE

1
 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

1.) Structural (chemical) elucidation

O NH O O
OH

‚ Natural product chemistry

O

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
‚ reaction kinetics product)
‚ 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-
‚ Structure Activity Relationships by NMR 13 complexed to a ligand
6.) Medicine -MRI

MRI images of the Human Brain

 Objectives
 Magnetic Shielding
 Chemical Shift
 Factors Affecting Chemical Shift
Inductive Effect by Electronegative Groups
s-character (Hybridization Effect)
Magnetic Anisotropic Effect
Hydrogen Bonding
 Equivalent and Nonequivalent Protons
 Integration of NMR Signal
 Spin-Spin Splitting (Spin-Spin Coupling)
4
 NMR History
First Observation of the Chemical Shift

H NMR spectra ethanol

1

Modern ethanol spectra

Arnold, J.T., S.S. Dharmatti, and M.E. Packard, J. Chem. Phys., 1951. 19: p. 507.
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
o I
2R

o – permeability of free space (4 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 (B) 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:

d 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:

1
H, 13C, 15N, 19F, 31P
biological and chemical relevance
Odd atomic mass
I = +½ & -½
NMR “inactive” Nuclear Spin (I) = 0:
12
C, 16O
Even atomic mass & number

Quadrupole Nuclei Nuclear Spin (I) >

½:
14
N, 2H, 10B
Even atomic mass & odd number
I = +1, 0 & -1
13
Gyromagnetic ratio ()

• magnetic moment is quantized (m)

m = I, I-1, I-2, …, -I

for common nuclei of interest:

m = +½ & -½

Isotope Net Spin  / MHz T-1 Abundance / %

1
H 1/2 42.58 99.98
2
H 1 6.54 0.015
3
H 1/2 45.41 0.0
31
P 1/2 17.25 100.0
23
Na 3/2 11.27 100.0
14
N 1 3.08 99.63
15
N 1/2 4.31 0.37
13
C 1/2 10.71 1.108
19
F 1/2 40.08 100.0
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:  =  Bo
/ 2
NMR Sensitivity

• Relative sensitivity of 1H, 13

C, 15
N and other nuclei NMR spectra
depend on
 Gyromagnetic ratio ()
 Natural abundance of the isotope

- Intrinsic property of nucleus can not be changed.
C)3 for 13C is 64x N)3 for 15N is 1000x

1
H 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 !!

1
H NMR spectra of caffeine 13
C NMR spectra of caffeine 13
C 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%20nois
 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 ≈ NS 1/2
 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
 MAGNETIC SHIELDING

 NMR would not be very valuable if all protons absorbed at the

same frequency.

 Different protons usually absorb different radiofrequencies (ν ).

 The electrons in a bond shield the nuclei from the magnetic

field. A moving charge (electron) creates a magnetic field, and
the field created by the moving electrons opposes the applied
magnetic field Bo.

 The electrons in a bond shield the nuclei from the magnetic

field.
 The more electron density around a proton, the more the
shield, the lower magnetic field affecting the proton ,
The less electron density , the less the shield , the
higher magnetic field.

This represents the electron density of a C-H bond. How much e

Z density is on the proton depends on what else is attached to th
C H is an elelctronegative atom, the carbon becomes electron defic
some of the electron density away from the H. if Z is an electro
group, more electron density ends up on the H.

25
 Shielded Protons
 The effective magnetic field, therefore, what the hydrogen nuclei actually
“sense” through the surrounding electronic environment is always less than the
actual applied magnetic field B0.

Beffective = Bapplied  Blocal

In the classical NMR experiment, magnetic field strength must be increased for 26
a
shielded proton to flip at the same frequency.
Low external applied field Higher external applied field

27
 Protons in a molecule

upfield

Lower frequency downfield

higher frequency

28
 The CHEMICAL SHIFT
The “chemical shift” is a field independent value.

Chemical shift is : the difference in frequency between the sample and

the standard over the operation frequency.
parts per
million

chemical shift in Hz
shift
=  = = ppm
spectrometer frequency in MHz

This division gives a number independent

of the instrument used.
A particular proton in a given molecule will always come at the same
chemical shift (constant value).
Of course, we don’t do any of this, it’s all done automatically by the
NMR machine.
 Tetramethylsilane (TMS)
CH3
H3C Si CH3
CH3
 TMS is added to the sample as internal standard.

 TMS protons are all identical, highly shielded providing a single

sharp peak always isolated from peaks of interest. The TMS was
assigned .

 Organic protons absorb downfield (to the left) of the TMS signal.

 TMS is inert , highly soluble in organic liquids and easily removed

from samples by distillation. 30
FACTORS AFFECTING CHEMICAL SHIFT

Four major factors account for the resonance

positions (on the ppm scale) of most protons

1. Deshielding by electronegative elements

Inductive effect by electronegative groups

2. s-character (hybridization effect)

3. Magnetic Anisotropic effect (magnetic fields usually due to -

bonded electrons in the molecule(

4. Hydrogen bonding.

31
 1. DESHIELDING BY ELECTRONEGATIVE
ELEMENTS

- 
Chlorine “deshields” the proton,

Cl C H that is, it takes valence electron

density away from carbon, which
-  in turn takes more density from
electronegative hydrogen deshielding the proton.
element

NMR CHART

“deshielded“ “highly shielded”

protons appear protons appear
at downfield at upfield
(higher ) (lower )

deshielding moves proton 32

resonance to lower field and higher 
 ELECTRONEGATIVITY DEPENDENCE OF
CHEMICAL SHIFT

Dependence of the Chemical Shift of CH3X on the Element X

Compound CH3X CH3F CH3OH CH3Cl CH3Br CH3I CH4 (CH3)4Si

Element X F O Cl Br I H Si
Electronegativity of X 4.0 3.5 3.1 2.8 2.5 2.1 1.8
Chemical shift  4.26 3.40 3.05 2.68 2.16 0.23 0

most TMS
deshielded
deshielding increases with the
electronegativity of atom X

33
 SUBSTITUTION EFFECTS ON CHEMICAL SHIFT

most
Cumulative Effect
The effect
deshielded increases with
CHCl3 CH2Cl2 CH3Cl
greater numbers
7.27 5.30 3.05 ppm of electronegative
atoms.

most
deshielded CH2Br CH2CH2Br X CH2CH2CH2Br
3.30 1.69 1.25 ppm

The effect decreases with increasing distance

from the electronegative atom. The effect
completely vanished at the fourth bond from the
electronegative atom. 34
2. s-CHARACTER
(HYBRIDIZTION OF CARBON
ATOM)

35
 As s-character of carbon atom increases, the electronic cloud is held more closer to
the carbon and provides less electron density for shielding of protons, and thus the
chemical shift, δ, increases (shifted downfield).
 According to the above reasoning, the following trend for the chemical shift is
expected:

C H > C H > C H
sp sp2 sp3
expected to be observed
at  > 7
s-character increases
(down field - higher )
 But in fact, we have the following order: actually observed
at  = 2-3

C H > C H > C H
sp2 sp sp3

 This suggested that other factors than the sp character of carbon might affect the
chemical shift in this case. The above discrepancy can be explained by what is
36
called Magnetic Anisotropic Effect.
3. MAGNETIC ANISOTROPIC FIELDS
DUE TO THE PRESENCE OF -BONDS

The presence of a nearby pi bond or pi system

greatly affects the chemical shift.

Induced magnetic fields due to the  - electrons have

greatest effect.

37
Aromatic protons
 = 7-8 ppm

38
Vinyl (Olefinic) protons,
 = 5-6 ppm

39
Acetylene protons
̃ ≈ 2.5 ppm

40
Aldehyde proton
= 9-10 ppm

Electronegative
oxygen atom

41
 4. HYDROGEN BONDING
O-H and N-H Signals
HYDROGEN BONDING DESHIELDS PROTONS
The chemical shift depends
R on how much hydrogen bonding
is taking place (observed in high
O H H concentrated solutions).
Hydrogen bonding lengthens the
O H O R O-H bond and reduces the valence
electron density around the proton
R
 it is deshielded and shifted
downfield in the NMR spectrum.

Alcohols vary in chemical shift D2O-exchangeable (peak for OH proton

from 0.5 ppm (free OH) to about in alcohol and NH in amines disappears
5.0 ppm (lots of H bonding). upon shaking with D2O) 42
 SOME MORE EXTREME EXAMPLES
δ- δ+ δ-
O H O
-I
Carboxylic acids have strong
R C R hydrogen bonding - they
C
form dimers.
O H O
δ- δ+ δ-
 Resonance, electronegativity of oxygen and the formation of hydrogen bonding
withdraw electron cloud from the acid protons.
 Thus, protons attached to carboxylic acids are the least shielded protons and
have a chemical shift of 10-12 ppm.

H3C O
O In methyl salicylate, which has strong
H
internal hydrogen bonding, the NMR
absorption for O-H is at about 14 ppm,
O
(highly downfield(

Notice that a stable 6-membered ring is formed 43

 NMR CORRELATION CHART
Chemical shift gives the electronic environment of protons
(Shielding and Deshielding)
PROTON IN PROTON IN
ELECTRON-POOR disappears upon shaking ELECTRON-RICH
ENVIRONMENTS with D2O ENVIRONMENTS
aromatic -OH -NH
DESHIELDED SHIELDED
DOWNFIELD UPFIELD
HIGHFREQUENCY CHCl3 , H 0 - 5 LOWFREQUENCY
LARGE  SMALL 
3 - 5
10-12 7-8 1.5 - 3
9-10 4.5 - 7 0 – 2 TMS

12 11 10 9 8 7 6 5 4 3 2 1 0  (ppm)
H
CH2F CH2Ar C-CH-C
RCOOH RCHO C=C CH2Cl CH2NR2
C
acid aldehyde olefinic CH2Br CH2S
C-CH2-C
sp2 CH2I C C-H
C=C-CH2 C-CH3
CH2O
CH23NO2 CH32-C- sp3 44
sp sp sp O
EQUIVALENT AND NONEQUIVALENT PROTONS
H
All of the protons in a molecule which are in chemically H H H2C CH2
identical environments will often exhibits the same
H2C CH2
chemical shift i.e., shows one signal in NMR spectrum at
H H C
the same value of . H2
H
O
The protons in this case are said to be (CH3)4Si
H3C CH3
chemically equivalent
Molecules giving rise to one NMR absorption signal
CH3 All protons are chemically equivalent
O
H H
H2C C O CH3
H2C C O CH3
H H
O On the other hand, molecules which have sets
CH3
of protons which are chemically distinct (have
O H different chemical environments) from one
CH3 H3C O C Cl another give rise to different absorption
H3C O
H signals from each other.
Molecules giving rise to two NMR absorption signals
Two dif ferent sets of chemically equivalent protons

Chemically nonequivalent protons 45

INTEGRATION OF A PEAK

Not only does each different type of hydrogen give a

distinct peak in the NMR spectrum, but we can also tell
the relative numbers of each type of hydrogen by a
process called integration.

Integration = determination of the area

under a peak
46
 • In the NMR spectrum, the area under each peak is proportional to the number of hydrogens
generating that peak.

• The NMR spectrometer has the capability to electronically integrate the area under each
peak.

 The integrated area measured by

2,2-dimethyl-1-propanol (C4H12O)
a ruler are
5 : 2.5 : 22

 Divide by the smallest number

give us the simplest ratio of 2 :
1:9

 Note that the integration gives

only ratios, not absolute values
for the number of the hydrogen
present in the sample
47
 Modern spectrometers
automatically print out the
integrals as numbers on the
spectrum

NMR spectrum can reveal the number of protons assigned for each signal

Integral lines : no. of protons in each signal 48

SPIN-SPIN SPLITTING

49
 SPIN-SPIN SPLITTING

Often a group of hydrogens will appear as a multiplet

rather than as a single peak.

Multiplets are named as follows:

Singlet (s) Quintet (quin)

Doublet (d) Sixtet (six)
Triplet (t) Septet (sept)
Quartet (q) Multiplet (m)

This happens because of interaction with neighboring

hydrogens and is called
SPIN-SPIN SPLITTING
Spin-spin coupling 50
 1,1,2-Tribromoethane

Nonequivalent protons on adjacent carbons.

Chapter 13 51
 THE ORIGIN OF
SPIN-SPIN SPLITTING

HOW IT HAPPENS

Spin-spin splitting arises because hydrogen on adjacent

carbon atoms can “sense” one another

52
 Doublet
1 adjacent proton

Chapter 13 53
 Triplet
2 Adjacent Protons

Chapter 13 54
 n + 1 RULE
MULTIPLETS
this hydrogen’s peak these hydrogens are
is split by its two neighbors split by their single
neighbor
singlet
doublet
H H H H triplet
quartet
C C C C quintet
H H sixtet
two neighbors
septet
n+1 = 3 one neighbor
triplet n+1 = 2
doublet
Where n is the number of EQUIVALENT protons on
Adjacent carbon atoms 55
 EXCEPTIONS TO THE n+1 RULE
IMPORTANT !

1) Protons that are equivalent by symmetry usually do not split one another

X CH CH Y X CH2 CH2 Y
no splitting if x = y splitting if x  y
two triplets
2) Protons in the same group usually do not split one another
H H
C H or C
H H
3) Splitting is not observed if the protons are separated by more than three 
bonds

56
INTENSITIES OF MULTIPLET PEAKS
PASCAL’S TRIANGLE
Intensities of
multiplet peaks 1 singlet

1 1 doublet

1 2 1 triplet

1 3 3 1 quartet

57
 NMR spectrum indicates the carbon skeleton
Spin-spin splitting gives the number of equivalent protons on
adjacent carbon atoms

58
Summary
INFORMATION WE CAN GET FROM NMR SPECTRUM
 Number of 1H-NMR signals = Number of kinds of protons.

 Chemical shift gives the electronic environment of protons

(Shielding and Deshielding).

 NMR spectrum can reveal the number of protons assigned for each
signal (integral lines : no. of protons in each signal).

 NMR spectrum indicates the carbon skeleton Spin-spin splitting gives

the number of equivalent protons on adjacent carbon atoms.

59
 Typical Values

Note that these are typical values and that there

are lots of exceptions!
60
Benzyl acetate
Cumene
Chemical Shift
Chemical Equivalence
Magnetic Equivalence
Coupling and Coupling Constant
Splitting pattern
Chemical Shifts and Multiplicities of Residual Protons in Commercially
Available Deuterated Solvents
C-13 NMR Spectroscopy
C-13 Spectrum of
m-Nitroacetophenone

O
CCH3

NO2
1H NMR Spectrum of
Ethylbenzene

CH2CH3
Typical ppm ()Ranges in
C-13 NMR
Relaxation Times
Coupling Constants are Large
J Values measured in Hz
HC CCH2CH2CH2CH3
HC CCH2CH2CH2CH3
 O O
CH3CH2OCCH2CCH3
 O O
CH3CH2OCCH2CCH3
H2C=C=CHCH2CH2OH
CO2H
H3C O
N CCH3
H3C
Silverstein, p. 212
Silverstein, p. 215
H3C OCH3
N CH
H3C OCH3
 O
H3C NHCCH3
C C

HC H
O
 O
CF3COCH3
Predicting Chemical Shifts
 Predicted Chemical Shifts of
Ca and Cb
b
a

Ca = (-2.5) + 4(9.1) + 9.4 + 2(-2.5) + 3(-1.5) + (-8.4) = 25.4 ppm

base 4o(1o) 4o(2o)

Cb = (-2.5) + 2(9.1) + 5(9.4) +(-7.2) + (-2.5) = 53.0 ppm

base 2o(4o) 2o(3o) 
Trimethylpentane-2,2,4

53.28, 31.10, 30.16, 25.51, 24.73

Chemical Shift
Prediction with
Functional Groups
Use Base Value From Table 4.5

Using Table 4.5 and 4.6

OH

OH
OH
34.7 + 41 = 75.7 22.8 + 41 = 63.8 13.9 + 48 = 51.9 ppm
 You can Choose Which Base
Value from Table 4.5 to Use
OH OH

OH OH

34.7 + 41 + (-5) + 3(9.4) + (-2.5) + (-7.2) = 89.2 ppm

C-3 -OH -OH 2o(3o) 2o(4o)
36.9 + 41 + (-5) + 2(9.4) + (-2.5) = 88.8 ppm
C-3 -OH -OH 2o(3o)
Spectrum at 75 MHz and 150 MHz
 Resources and references
Textbook: Principles of Instrumental Analysis, Skoog,
Holler, Nieman

Recommended further reading:

“Principles of instrumental analysis, 5th ed. by Skoog, Holler,
Nieman” Chapter 19.

Extra resources are available on the intranet.

Relevant web sites

http://www.chemguide.co.uk/analysismenu.html

108