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NMR Spectroscopy
Guangjin Hou
22-04-2009
NMR History
1H
NMR spectra of water
(First NMR Spectra on Water, 1946)
1H
NMR spectra ethanol
(First Observation of the
Chemical Shift, 1951)
NMR History
Modern ethanol spectra
1937
1946
1953
1966
1975
1985
1987
1990
1996/7
……
Rabi predicted NMR
Bloch, Purcell first NMR of bulk sample
Overhauser NOE (nuclear Overhauser effect)
Ernst, Anderson Fourier transform NMR
Jeener, Ernst 2D NMR
Wüthrich first solution structure of a small protein (BPTI)
from NOE derived distance restraints
3D NMR + 13C, 15N isotope labeling of recombinant proteins (resolution)
pulsed field gradients (artifact suppression)
new long range structural parameters:
- residual dipolar couplings from partial alignment in liquid crystalline media
- projection angle restraints from cross-correlated relaxation TROSY
(molecular weight > 100 kDa)
NMR (Nuclear Magnetic Resonance)
NMR measures the absorption of electromagnetic radiation in the radio-frequency
region (~4-950 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 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!
Theory of NMR
Faraday’s Law
= electron
= neutron
= proton
A moving perpendicular external magnetic
field will induce an electric current in a
closed loop
An electric current in a closed loop will
create a perpendicular magnetic field
Theory of NMR
l
Quantum Description
Nuclear Spin
•Nucleus rotates about its axis (spin)
•Nuclei with spin have angular momentum (p) or spin
•
quantized, spin quantum number I
•
2I + 1 states: I, I-1, I-2, …, -I
•
identical energies in absence of external magnetic field
When placed in a magnetic field of strength B0, the energy for different
states would be different, i.g. 1H (I=1/2), there are +½, -½ states, the energy
difference is (γ, gyromagnetic ratio),
ΔE = h ⋅ γ ⋅ B0
Note: NMR cannot detect “inactive” Nuclear Spin (I) = 0, e.g.
12C, 16O. Fortunately, almost every element in the periodic
table has an isotope with a non zero nuclear spin.
NMR Signal
B0
Magnetic alignment
−
M
Bo > 0
Bo = 0
+
NMR Sensitivity
Strength of the NMR signal depends on the population difference between
the α and β spin states, and the population (N) difference can be
determined from the Boltzmman distribution and the energy separation:
N−
+
= e −ΔE / kT
N
e.g. 1H at 400 MHz (Bo = 9.39 T) : ΔE = 6 x 10-5 Kcal / mol
N+/N- = 1.000060
To enhance NMR signal:
S ∝ γ 5/ 2 B03/ 2 NB12 g (ν ) / T
1) Number of Nuclei (N) (quantity of the sample) (limited to field
homogeneity and filling factor)
2) Gyromagnetic ratio (in practice γ5/2)
3) Inversely to temperature (T)
4) External magnetic field (Bo3/2, in practice, homogeneity)
5)
B12 exciting field strength (RF pulse)
NMR Sensitivity
To Increase Magnet Strength is a Major Means
HSQC@600M
HSQC@900M
NMR Sensitivity
To Increase Magnet Strength is a Major Means
$800,000
$2,000,000
$5,000,000
NMR spectrometers
Three Main Series
Varian INOVA
The first 40 MHz NMR spectrometer
(Varian, 1953)
The first 90 MHz FTNMR
spectrometer (Bruker, 1967)
Bruker AVANCE
JEOL ECA
Information in NMR Spectra
chemical shift
splitting
linewidth
Observable
Name
Quantitative
Information
Peak position
Chemical shifts (δ)
δ(ppm) = υobs –υref/υref (Hz)
chemical (electronic)
environment of nucleus
Peak Splitting
Coupling Constant (J) Hz
peak separation
(intensity ratios)
neighboring nuclei
(torsion angles)
Peak Intensity Integral
unitless (ratio)
nuclear count (ratio)
relative height of integral curve T1 dependent
Peak Shape
Δυ = 1/πT2
peak half-height
Line width
molecular motion
chemical exchange
uncertainty principal
uncertainty in energy
Development of Multi-Dimensional NMR
Developing MD NMR in order to
1. Spread spectrum out
– resolve peaks
– remove overlap
2. Supply more information
……
1967
1971
1987
1990
……
Richard Ernst
Jean Jeener
Griesinger et al.
Ad Bax et al.
- Fourier transformations
- Two dimensional NMR - COSY
- Advent of three dimensional NMR
- Four dimensional NMR
NMR structure of Taxol
(anticancer drug )
O
Typical Applications of NMR
O
O
NH
O
O
OH
O
OH
HO
O
O
1.) Structural (chemical) elucidation
™Natural product chemistry
NMR structure of protein™Synthetic chemistry
ligand complex
- analytical tool of choice of synthetic chemists
- used in conjunction with MS and IR
2.) Study of dynamic processes
™reaction kinetics
™study of equilibrium (chemical or structural)
3.) Structural (three-dimensional) studies
™Proteins, Protein-ligand complexes
MRI images of human brain
™DNA, RNA, Protein/DNA complexes
™Polysaccharides
4.) Drug Design
Structure Activity Relationships by NMR
5) Medicine -MRI
O
O
O
3D Structure determination by NMR
Drug Discovery by NMR
Structure Activity Relationships, SAR
15
N(ppm)
¾ Proposed by the Abbott Laboratories (Fesik
et al, 1996)*
¾ NMR method of screening and optimization
of small organic molecules that bind the
protein
¾ Based on chemical shift perturbation upon
substrate binding/2D Correlation Spec
¾ Good for eliminating false positives
¾ Non-overlapping ligands can potentially be
linked to form a more effective (nM)
compound
1
H(ppm)
Dynamic investigation by NMR
NMR is a convenient way to study rate of reactions or motions, provided that the
lifetime of participating species is comparable to appropriate scales
ps
relaxation
T1
ns
μs
CMPG
T1ρ, T2
ms
s
min, hours …
2D exchange
H/D exchange
Summary on application of NMR spectroscopy
NMR has extensively applied into various
fields including:
‰ from gas to liquid, to solid
‰ from inorganic to organic
‰ from material to biology, to medicine
‰ from structure to dynamics
‰ form physical change to chemical
reaction to biological mechanism
‰ ……
Other NMR Facilities
¾
¾
¾
¾
¾
¾
¾
NMR imaging spectrometer
NMR Teslameter 、Gaussmeter
Magnetic Field Mapping system
NMR Analyzer
NMR-MOUSE (MObile Universal Surface Explorer)
NMR water explorer
NMR logging instrument
e.g.
Thanks for your attention!
The end