Modern NMR techniques for chemical structure

elucidation
-ethanol and beyond...
Dr. Tim Claridge
tim.claridge@chem.ox.ac.uk

Introduction
Historical overview
NMR Instrumentation

Introducing NMR spectroscopy

NMR techniques for structure elucidation
Undergraduate NMR course content

What is NMR spectroscopy?

Nuclear- dealing with the property of nuclear spin
Magnetic- interaction of nuclear spins with applied magnetic fields
Resonance Spectroscopy- excitation of these nuclear spins

Why do we use it?

Molecular structure
conformation

dynamics
interactions

Nuclear Spin
spin

B0

precession

External
magnetic
field

magnetic moment

and Resonance
N
External
magnetic
field

DE

Electromagnetic pulse of energy DE

DE = hn

History: the beginning

1946: Nuclear Magnetic Resonance
(Nuclear induction)
Purcell, Torrey and Pound- 1 kg Paraffin wax
Bloch, Hansen and Packard- 850 ml water

Organic NMR spectroscopy

1951: First published high-resolution NMR spectrum:
Neat ethanol @ 30 MHz

HO-CH2-CH3

And now
10 mg incubation product from antibiotic biosynthesis
pathway (700 MHz, cryogenic probe)
Me
HO 2C

N
H

CO2 H

NMR instrumentation

robot

console

preamplifier

Magnet
400 MHz
anti-vibration
platform

Magnet development
B0 /Tesla

1000

23.5
1H Frequency (MHz)

21.1
800

18.8
16.4
14.1

600

20 MHz/year

11.7
400

8.5
7.0

200

0
1950

1960

1970

1980

Year

1990

2000

2010

Superconducting magnet

NMR probeheads

Radiofrequency
transmit & receive
coils

Tuning circuitry

Cryogenic probeheads
Cold head

Sample @ ~ 300K

Probe detection coils @ ~25K

NMR signal preamplifier @ ~ 70K

Thermal noise reduced significantly

Introducing NMR Spectroscopy

The electromagnetic spectrum

Hydrogen NMR spectrum (1H)

3) Absorption intensities:
Peak integration

2) Spin-coupling
fine structure
CH3
H2C

8.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

1) Chemical shift

3.0

2.5

2.0

1.5

1.0

0.5 ppm

Introducing NMR Spectroscopy

Features of the NMR Spectrum

1. Chemical shifts
Tell us about the local chemical environments of each nucleus

2. Spin-spin couplings

Tell us about the interactions of each nucleus with its

neighbouring nuclei

3. Peak intensities (integrals)

Tell us the relative number of nuclei in each chemical

environment

Feature I: Chemical Shifts (d)

Chemical shifts reflect chemical environments: nuclei act as

undercover spies reporting on their surroundings
1H

Spectrum

9.5

9.0

8.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

frequency

3.0

2.5

2.0

1.5

1.0

ppm

The origin of chemical shifts

Electron spins
surround nucleus

External
magnetic
field

their own
magnetic fields
shield nucleus
from external field
..differences in
electronic shielding
produce differences
in chemical shifts

Feature II: Spin Spin couplings (J)

A nucleus can sense the presence of its neighbours
across bonds: nuclear spies again
Strength of interaction reflected in the spin-spin coupling
constant, J (Hz)

Bond to HQ. Come in HQ

Im next to three hydrogensout!

and Im next to two hydrogensout!

CH3
H2C

2.9

2.8

2.7

2.6

2.5

2.4

2.3

2.2

2.1

2.0

1.9

1.8

1.7

1.6

1.5

1.4

1.3

1.2

ppm

Spin coupling patterns and multiplicities

doubledoublet (dd)

doublet (d)

triplet (t)
double
triplet (dt)

triple
doublet (td)

Feature III: Resonance intensities

Total resonance intensity reflects the number of nuclei in
that environment: the number of spies present
Calculated by integration of peak areas

CH3
H2C

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

ppm

NMR techniques
for chemical structure elucidation

NMR

NMR in chemical research

BnO
CH 2
H

BnO

Hydrogen

(1H)

NMR spectra

BnO

O
H
H

OBn
H

CH 2
H

BnO
BnO

STol
H
H

OBn
H

Multi-pulse NMR- spin gymnastics

Carbon NMR and editing

12C

is NMR inactive,

13C

is NMR active but only 1% abundant

Standard carbon spectra hide multiplicity information (eg CH vs CH2 vs

CH3)- simple stick appearance
Chemical shifts indicate chemical environments
Carbon editing experiments indicate protonation state

Two-dimensional (2D) NMR Spectroscopy

Maps or correlates nuclear interactions within molecules:
Through-bond coupling (J)
Indicates presence of bonding connectivity
Through-space coupling (nOe)
Indicates close spatial proximity -> 3-dimensional shapes of molecules
& stereochemistry

2D as two frequency axes

1D
8.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5 ppm

2D contour presentation

Top-down view: Contour plot

1H-1H

2D
Correlation
Spectroscopy (COSY)
Maps spin-spin coupled partners

H2N

CH

CH

OH

OH

CH3

Threonine
ppm

coupled hydrogen
spin system

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5

5
ppm

2D COSY

2D 1H-1H Total Correlation Spectroscopy (TOCSY)

Maps all spin-spin interactions within molecular fragments or even complete molecules

TOCSY

COSY

F1

F2

F1

F2

2D TOCSY

Heteronuclear correlation methods:

1H-13C
A
H
H

2
3

H C
H

2D Heteronuclear correlation

13C

1H

Biological chemistry:
Peptides of 19 amino-acids

Chemical biology:
Protein of 110 amino acids

1H-15N

correlation: 110 amino acids

Isotopically 15N labelled protein

Structure
Drug binding

Protein-protein
interactions

15N

1H

3D NMR methods
for protein structures:

triple-resonance NMR 1H/13C/15N

HNCA

C
N

C
C

HNCO

HNCACO

C
C

15N

15N

13C

1H

1H

13C/15N

labelled
Protein

Medicine:
Magnetic Resonance Imaging

Functional MRI Unit, Oxford

Undergraduate NMR spectroscopy

Often part of Organic Spectroscopy courses (+UV, IR, MS)

YEAR

Material

2nd

1H

& 13C chemical shifts, spin-spin couplings, resonance intensities

Origins of chemical shifts and couplings
Correlating chemical shifts with structural environments
Correlating spin-spin couplings with structural features
Influences on NMR spectra: stereochemistry, conformation, dynamics
Properties of other NMR active nuclei: 19F, 31P, 11B
Instrumentation & Fourier transform NMR

3rd

Through-space correlations: nuclear Overhauser effects

defining stereochemistry and 3D shapes of molecules
2D NMR methods
1H-1H through-bond correlations
1H-13C through-bond correlations
1H-1H through-space correlations
Physical behaviour of nuclear spins: relaxation

Thank you for your attention

tim.claridge@chem.ox.ac.uk