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NMR Techniques
Citation preview
Modern NMR techniques for chemical structure
elucidation
-ethanol and beyond...
Dr. Tim Claridge
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
magnetic moment
precession B0
External magnetic
field
N
S
and Resonance
DE
a
b S
N External magnetic
field
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)
NH
HO2C
CO2H
Me
NMR instrumentation
Magnet 400 MHz
robot
console
anti-vibration platform
preamplifier
0
200
400
600
800
1000
1950 1960 1970 1980 1990 2000 2010
1H
Fre
qu
en
cy (
MH
z)
Year
20 MHz/year
Magnet development
11.7
14.1
16.4
18.8
21.1
8.5
7.0
B0 /Tesla
23.5
Superconducting magnet
NMR probeheads
Radiofrequency transmit & receive
coils
Tuning circuitry
Cryogenic probeheads
Probe detection coils @ ~25K
NMR signal preamplifier @ ~ 70K
Sample @ ~ 300K
Thermal noise reduced significantly
Cold head
Introducing NMR Spectroscopy
The electromagnetic spectrum
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
Hydrogen NMR spectrum (1H)
H2C
CH3
1) Chemical shift
2) Spin-coupling fine structure
3) Absorption intensities: Peak integration
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
Chemical shifts reflect chemical environments: nuclei act as
undercover spies reporting on their surroundings
Feature I: Chemical Shifts (d)
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 3.0 2.5 2.0 1.5 1.0 ppm
1H Spectrum
frequency
The origin of chemical shifts
+
N External magnetic
field
Electron spins surround nucleus 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)
1.21.31.41.51.61.71.81.92.02.12.22.32.42.52.62.72.82.9 ppm
H2C
CH3
Bond to HQ. Come in HQ Im next to three hydrogensout!
and Im next to two hydrogensout!
J
Spin coupling patterns and multiplicities
doublet (d) double-
doublet (dd)
triplet (t)
triple doublet (td)
double triplet (dt)
1.52.02.53.03.54.04.55.05.56.06.57.07.5 ppm
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
H2C
CH3
5 2 3
NMR techniques
for chemical structure elucidation
NMR
NMR in chemical research Hydrogen (1H) NMR spectra
O
H
BnO
H
BnO
H
H
OBnH
CH2
BnO
O
H
O
H
BnO
H
H
OBnH
CH2
STol
BnO
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
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
1D
2D contour presentation
Top-down view: Contour plot
ppm
0.51.01.52.02.53.03.54.04.5 ppm
5
4
3
2
1
H2N CH C
CH
OH
O
OH
CH3
Threonine
coupled hydrogen
spin system
2D 1H-1H Correlation
Spectroscopy (COSY)
Maps spin-spin coupled partners
2D COSY
2D 1H-1H Total Correlation Spectroscopy (TOCSY)
Maps all spin-spin interactions within molecular fragments or even complete molecules
A B C D
F2
F1
COSY
A B C D
F2
F1
TOCSY
2D TOCSY
HH
H
H
A
B
C
D
12
3
Heteronuclear correlation methods: 1H-13C
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
1H
15N
Structure Drug binding Protein-protein interactions
3D NMR methods
for protein structures: triple-resonance NMR 1H/13C/15N
HNCA
1H
15N
1H
15N
13C
N
C
C
N
H
H
C
C
O
O
N
C
C
N
H
H
C
C
O
O
N
C
C
N
H
H
C
C
O
O
HNCACO HNCO
N
C
C
N
H
H
C
C
O
O
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