L
ECTURE
COURSE: NMR S
PECTROSCOPY
1
Table of Content
The physical basis of the NMR experiment
5
The Bloch equations: 8
Quantummechanical treatment: 9
The macroscopic view: 10
Fourier Transform NMR: 14
The interaction between the magnetization and the additonal
RF (B1) field: 14
Description of the effect of the B1 field on transverse and lon
gitudinal magnetization using the Bloch equations: 16
The excitation profile of pulses: 17
Relaxation: 18
The intensity of the NMR signal: 20
Practical Aspects of NMR:
The components of a NMR instrument
The magnet system: 22
The probehead: 23
The shim system: 25
The locksystem: 28
The transmitter/receiver system: 28
Basic data acquisition parameter 31
Acquisition of 1D spectra 36
Calibration of pulse lengths: 36
Tuning the probehead: 38
Adjusting the bandwidth of the recorded spectrum: 39
Data processing:
40
Phase Correction 43
Zerofilling and the resolution of the spectrum 46
Resolution enhancement and S/N improvement 47
Exponential multiplication: 48
LorentztoGauss transformation: 48
SineBell apodization: 49
Baseline Correction 50
Linear prediction: 51
The chemical shift:
53
The diamagnetic effect: 53
The paramagnetic term: 54
Chemical shift anisotropy: 56
Magnetic anisotropy of neighboring bonds and ring current
shifts: 57
Electric field gradients: 59
Hydrogen bonds: 59
Solvent effects: 60
Shifts due to paramagnetic species: 60
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2
Scalar couplings:
60
Direct couplings (1J): 62
Geminal couplings (2J): 63
Vicinal couplings (3J): 63
Longrange couplings: 65
Couplings involving p electrons: 65
The number of lines due to scalar spin,spin couplings: 65
Strong coupling: 67
Relaxation:
68
T1 relaxation: 68
T2 relaxation: 69
The mechanisms of relaxation: 70
Other relaxation mechanisms: 72
Chemical shift anisotropy (CSA): 72
Scalar relaxation: 72
Quadrupolar relaxation: 73
Spinrotation relaxation: 73
Interaction with unpaired electrons: 73
The motional properties: 73
The dependence of the relaxation rates on the fluctuating fields
in x,y or z direction: 75
Excurs: The LipariSzabo model for motions: 77
The nature of the transitions: 78
Measurement of relaxation times: 80
The Nuclear Overhauser Effect (NOE):
84
Experiments to measure NOEs: 86
The steadystate NOE: 87
Extreme narrowing (hmax >0): 87
Spindiffusion (hmax <0): 88
The transient NOE: 89
The state of the spin system and the density matrix: 90
The sign of the NOE: 92
Why only zero and doublequantum transitions contribute to
the NOE 94
Practical tips for NOE measurements: 96
Chemical or conformational exchange:
99
Twosite exchange: 99
Fast exchange: 101
The slow exchange limit: 102
The intermediate case: 102
Investigation of exchange processes: 103
EXSY spectroscopy: 103
Saturation transfer: 104
Determination of activation parameters: 105
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3
The product operator formalism (POF) for description of pulseexperiments:
106
RF pulses: 107
Chemical shift precession: 107
Scalar spin,spin coupling: 108
A simple onedimensional NMR experiment: 110
The effect of 180 degree pulses: 111
Coherence transfer: 112
Polarization transfer: 113
TwoDimensional NMR Spectroscopy:
118
The preparation period: 120
The evolution period: 121
The mixing period: 122
The detection period: 124
Hetcor and inversedetection experiments: 124
Phasecycling: 125
An Alternative: Pulsed Field Gradients 126
Hybrid 2D techniques: 128
Overview of 2D experiments: 129
Original references for 2D experiments: 130
Solid State NMR Spectroscopy:
132
The chemical shift 132
Dipolar couplings: 135
Magic Angle Spinning (MAS) 136
Sensitivity Enhancement: 136
Recoupling techniques in SSNMR: 137
SSNMR of oriented samples: 140
Labeling strategies for solidstate NMR applications: 142
L
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COURSE: NMR S
PECTROSCOPY
4
A
PPLICATION
F
IELDS
OF
NMR S
PECTROSCOPY
Highresolution NMR spectroscopy
Analytics
"small" molecules
determination of the covalent structure
determination of the purity
Elucidation of the 3D structure
"small" molecules
determination of the stereochemistry: cis,trans isomerism,
determination of optical purity
Biopolymers up to about 2030 kDa
determination of the 3D solution structure
provided
the pri
mary sequence is known!
investigation of the interaction of molecules (complexes)
investigation of the dynamics of proteins
1
H,
13
C or
15
N relaxation measurements
1
H,
1
H oder
1
H,
15
N NOE measurements
Determination of the kinetics of reactions
Solidstate NMR spectroscopy
insoluble compounds (synthetic polymers)
very large compounds (requires speciﬁc labels)
determination of the structure in the solidstate (vs. liquidstate)
determination of the dynamics in the solidstate
Imaging techniques
spin tomography
"In vivo" NMR spectroscopy
distribution of metabolites in the body
L
ECTURE
C
OURSE
: NMR S
PECTROSCOPY
First Chapter: Physical Basis of the NMR Experiment Pg.5
1. T
HE
PHYSICAL
BASIS
OF
THE
NMR
EXPERIMENT
Imagine a charge travelling circularily about an axis. This is similar to a current
that ﬂows through a conducting loop:
Such a circular current builds up a magnetic moment
µ
whose direction is per
pendicular to the plane of the conducting loop. The faster the charge travels
the stronger is the induced magnetic ﬁeld. In other words, a magnetic dipole
has been created.
Such dipoles, when placed into a magnetic ﬁeld, are expected to align with the
direction of the magnetic ﬁeld. In the following we will look at a mechanical
equivalent represented by a compass needle that aligns within the gravita
tional ﬁeld:
When such a compass needle is turned away from the northpole pointing
direction to make an angle
φ
a force acts on the needle to bring it back. For the
case of a dipole moment that has been created by a rotating charge this force is
proportional to the strength of the ﬁeld (
B
) and to the charge (
m
).
The
torque
that acts to rotate the needle may be described as
in which
J
is deﬁned as the
angular momentum
which is the equivalent for rota
FIGURE 1.
FIGURE 2.
µ
N
φ
T
t ∂
∂J
r F × = =
L
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: NMR S
PECTROSCOPY
First Chapter: Physical Basis of the NMR Experiment Pg.6
tional movements of the linear momentum.
Note that the direction of the momentum is tangential to the direction along
which the particle moves. The torque is formed by the vector product between
the radius and the momentum (see additional material) and is described by a
vector which is perpendicular to both radius and momentum. In fact, it is the
axis of rotation which is perpendicular to the plane. The corresponding poten
tial energy is
In contrast to the behaviour of a compass needle the nuclear spin does not
exactly align with the axis of the external ﬁeld:
FIGURE 3. Left: linear momentum. Right: angular momentum
FIGURE 4. Rotation of the nuclear momentum about its own axis (blue) and about the magnetic ﬁeld axis (red).
p = m v
J = r x p
Excurse: Corresponding parameter for translational and rotational movements
PureTranslation (fixed direction) Pure Rotation (fixed axis)
Position x θ
Velocity v = dx/ dt ω =dθ/ dt
Acceleration a = dv/ dt α = dω/ dt
Translational (Rot.) Inertia m I
Force (Torque) F T = r x F
Momentum p = mv J = r x p
Work W = Int F dx W = Int T dθ
Kinetic energy
K = 1/ 2 mv
2
K = 1/2 Iω
2
Power P = F v P = Τ ω
E
pot
T ϕ d
0
ϕ
∫
– =
B
ω
LECTURE COURSE: NMR SPECTROSCOPY
First Chapter: Physical Basis of the NMR Experiment Pg.7
This is a consequence of its rotation about its own axis. This property is called
spin. It rotates (spins) about its own axis (the blue arrow) and precesses about
the axis of the magnetic ﬁeld B (the red arrow). The frequency of the precession
is proportional to the strength of the magnetic ﬁeld:
ω = γ B
The proportionality constant is called the gyromagnetic ratio.
The frequency ω is expressed in terms of a angular velocity (see additional
material). It is speciﬁc for the kind of nucleus and therefore has a different
value for
1
H,
13
C,
19
F etc. The precession frequency
ω
0
= ν
ο
2 π
is called the Lamor frequency. In contrast to a compass needle which behaves
"classically" in the way that it can adopt a continous band of energies depend
ing only on the angle φ it makes with the ﬁeld the corresponding angle φ of the
nuclear dipole moment is quantitized. Hence, we will later introduce the quan
tummechanical treatment shortly.
Of course, we do not observe single molecules but look at an ensemble of mol
ecules (usually a huge number of identical spins belonging to different mole
cules). The sum of the dipole moments of identical spins is called
magnetization:
Excurse: The movement of a classical gyroscope
Imagine a wheel ﬁxed to a shaft. When the this gyroscope is placed with the shaft perpendicular to the
ground and released it will fall down (see Fig. below, left side). However, when the wheel spins about
the axis of the shaft, the gyroscope precesses about the axis perpendicular to the ground with a fre
quency ω that is called the precession frequency (right side) and takes a welldeﬁned angle φ with
respect to the rotation axis:
ω
φ
M µ
i
j
∑
=
LECTURE COURSE: NMR SPECTROSCOPY
First Chapter: Physical Basis of the NMR Experiment Pg.8
1.1 The Bloch equations:
The Bloch’s equations describe the fate of magnetization in a magnetic ﬁeld.
We have stated before that a force ( a torque) acts on a dipole moment when it
is placed inside a mgnetic ﬁeld such that the dipole moment will be aligned
with the direction of the static magnetic ﬁeld. Mathematically this is decribed
by forming the vector product between dipole moment and magnetic ﬁeld (see
add. material for the mathematics involved):
Considering that (see page 2)
and that
we ﬁnd that
which describes the timeevolution of the magnetization.
In the absence of an additional B
1
ﬁeld the components of the ﬁeld along the
cartesian axes are:
B
x
= 0
B
y
= 0
B
z
= B
o
which leads to the following set of coupled differential equations:
In order to drive the system into the equilibrium state (no transverse coher
ence, relative population of the α/ β states according to the Boltzmann distri
bution) additional terms have been phenomenologically introduced such as
M
x
/ T
2
for the M
x
component.
T M B × =
T
t ∂
∂J
=
M µ
i
i
∑
γ J
i
i
∑
= =
t ∂
∂M
γ
t ∂
∂
J γT γ M B × ( ) = = =
t ∂
∂
M
x
t ( )
γ M
y
B
0
M
x
T
2
t ∂
∂
M
y
t ( )
γ – M
x
B
0
M
y
T
2
⁄ –
=
⁄
t ∂
∂
M
z
t ( ) M
z
M –
0
( ) – T
1
⁄ =
–
=
LECTURE COURSE: NMR SPECTROSCOPY
First Chapter: Physical Basis of the NMR Experiment Pg.9
The solutions to these equations are given by:
The ﬁrst two equations describe mathematically a vector that precesses in a
plane (see add. mat.) and hence give the correct description for what we will be
looking at in a rather pictorial way in the following.
1.2 Quantummechanical treatment:
The dipole moment µ of the nucleus is described in quantummechanical terms
as
Therein, J is the spin angular momentum and γ the gyromagnetic ratio of the
spin. When looking at single spins we have to use a quantummechanical treat
ment. Therein, the zcomponent of the angular momentum J is quantitized
and can only take discrete values
where m is the magnetic quantum number. The latter can adopt values of m = 
I, I+1, …,0,...,I1, I
with I being the spin quantum number.
For I=1/ 2 nuclei, m can only be +1/ 2 or 1/ 2, giving rise to two distinct energy
levels. For spins with I=1 nuclei three different values for J
z
are allowed:
FIGURE 5. Vectorial representation of the zcomponent of the angular momentum for a spin I=1/2 (left) and spin
I=1 (right) nucleus.
M
x
t ( ) M
x
0 ( ) ωt cos M
y
– 0 ( ) ωt sin [ ]e
t T2 ⁄ – ( )
M
y
t ( ) M
x
0 ( ) ωt sin M
y
0 ( ) ω cos t + [ ]e
t T2 ⁄ – ( )
M
z
t ( ) M
eq
M
z
0 ( ) M
eq
– [ ] + e
t T1 ( ) ⁄ – ( )
=
=
=
µ = γ J
J
z
= m
h
2π
H
z
I=1
J
z
= +
J
z
= 
h
J
z
= 0
4π
h
4π
H
z
I=1/2
J
z
= +
J
z
= 
h
4π
h
4π
LECTURE COURSE: NMR SPECTROSCOPY
First Chapter: Physical Basis of the NMR Experiment Pg.10
The energy difference ∆E
pot
, which corresponds to the two states with m=±1/
2, is then
(the quantummechanical selection rule states, that only transitions with ∆m=
±1 are allowed):
1.3 The macroscopic view:
The NMR experiment measures a large ensemble of spins derived from a
huge number of molecules. Therefore, we now look at the macroscopic bevav
iour. The sum of the dipole moments of all nuclei is called magnetization. In
equilibrium the spins of I=1/ 2 nuclei are either in the α or βstate and precess
about the axis of the static magnetic ﬁeld. However, their phases are not corre
lated. For each vector pointing in one direction of the transverse plane a corre
sponding vector can be found which points into the opposite direction:
FIGURE 6. Energy levels of the α and β states of I=1/ 2 nuclei
FIGURE 7. Equilibrium state with similarily populated α and βstates (left), uncorrelated phases (middle) and no
net phase coherence (right).
E
pot
= µ
z
B = γ J B
α
β
B
o
= 0 B
o
≠ 0
Ε
∆E=hν
+ 1/ 2 γ h/ 2π B
 1/ 2 γ h/ 2π B
H
z
m= +1/ 2
m=  1/ 2
J
z
= +
J
z
= 
h
h
4π
4π
B
o=z
y
x x
y
x
y
Σ
B
o=z
y
x x
y
x
y
Σ
E
pot
= γ
h
2π
B
LECTURE COURSE: NMR SPECTROSCOPY
First Chapter: Physical Basis of the NMR Experiment Pg.11
Therefore, the projection of all vectors onto the x,y plane (the vector sum of the
transverse components) is vanishing provided that the phases of the spins are
uncorrelated.
However, application of a radiofrequency (RF) ﬁeld perpendicular to the mag
netic ﬁeld (e.g. along the x or yaxis), the socalled B
1
ﬁeld, creates a state in
which the phases of the spins are partially correlated. This state is called coher
ence. When projecting the vectors onto the x,y plane the resulting transverse
magnetization is nonvanishing giving rise to a signal in the detection coil.
When the motions of spins are described in vector diagrams most frequently
only the vector sum of the spins is shown in order to simplify it.
The magnitude and direction of the magnetization vector can be calculated by
vectorial addition of the separate dipole moments.
This is shown in the following ﬁgure in which the vector sum of longitudinal
(blue) and transverse (yellow) magnetization of uncorrelated spins which are
only in the αstate (A) or in both states (B) is displayed as well as for correlated
states (C and D): It is evident that only for correlated states transverse magneti
zation (magnetization in the x,y plane) is observed. Only transverse magneti
zation leads to a detectable signal in the receiver coil and hence contributes to
the NMR signal. Therefore, only the vector sum of the transverse component is
FIGURE 8. Coherent state of spins in α or β(left) states and the projection onto the x,y plane(middle) and sum
vector of the x,y component.
B
o=z
y
x
x
y
x
y
Σ
LECTURE COURSE: NMR SPECTROSCOPY
First Chapter: Physical Basis of the NMR Experiment Pg.12
shown to describe the relevant part of the spins:
The experiment setup of the spectrometer includes a radiofrequency coil,
which delivers the orthogonal B
1
ﬁeld. Simultaneously this coil serves to pick
up the NMR signal. To understand how the magnetization that rotates in the
transverse (x,y) plane induces the NMR signal it is convenient to look at the
vector sum of the transverse components which present a magnetic ﬁeld that
rotates in space:
The magnitude of the current that is induced in the receiving coil depends on
the orientation of the magnetization vector with respect to the coil. When the
FIGURE 9. Different states of the spin system (see text).
FIGURE 10. Spins precessing at different velocities (and hence have different chemical shifts) are color coded.
B
o=z
y
x
B
o=z
y
x
B
o=z
y
x
B
o=z
y
x
B
o=z
y
x
B
o=z
y
x
B
o=z
y
x
B
o=z
y
x
A
C
B
D
z
y
x
z
y
x
z
y
x
∆t
2∆t
LECTURE COURSE: NMR SPECTROSCOPY
First Chapter: Physical Basis of the NMR Experiment Pg.13
magnetization is pointing towards the coild the induced current is at maxi
mum. Because the magnetization rotates the induced current follows a sine (or
cosine) wave (see additional material). Spins with different chemical shift, dif
ferent larmor frequencies, precess at different rates and hence the frequency of
the current is the larmor frequency, e.g. the frequency of the precessing spins: .
In the picture above the induced current is shown for different orientations of
the transverse magnetization.
This situtation is very similar to a conducting loop that rotates in a magnetic
ﬁeld as encountered in a generator:
However, in the generator the (coil) loop is rotating and the magetic ﬁeld is
stationary, opposite to the situation in a NMR experiment. The amplitude of
the induced current is following a (co) sine wave. Similarily, the rotating
dipoles in NMR create a sinemodulated current.
FIGURE 11. Left:Rotating spin with its position at certain time intervals 16 are marked. Right: Corresponding
signal in the receiver coil.
FIGURE 12. Left: Conducting loop rotating in a magnetic ﬁeld with corresponding current induced (right).
z
y
x
1
2
3
4
5
6
I
Det
t
1
2
3
4
5
6
T
I
t
N
S
LECTURE COURSE: NMR SPECTROSCOPY
First Chapter: Physical Basis of the NMR Experiment Pg.14
1.4 Fourier Transform NMR:
Spins that belong to nuclei with different chemical environment precess with
different frequencies. For more complex compounds that contain many differ
ent spins the signal in the receiver coil is a superposition of many different fre
quencies. The Fourier Transformation is a convenient mathematical tool for
simultaneous extraction of all frequency components. It allows to transform
data from the time into the frequency domain:
In reality, the magnetization does not precess in the transverse plane for inﬁ
nite times but returns to the zaxis. This phenomenon is called relaxation and
leads to decreasing amplitude of signal in the detector with time.
1.5 The interaction between the magnetization and the additonal RF (B
1
) ﬁeld:
When only the static B
0
ﬁeld is present the spins precess about the zAxis (the
axis of the B
0
ﬁeld). To create spincoherence an additional RF ﬁeld is switched
on, that is perpendicular to the axis of the static ﬁeld (the socalled B
1
ﬁeld). To
emphazise that this ﬁeld is turned on for only a very short period of time usu
ally it is called a (RF) pulse. During the time where B
0
and B
1
ﬁeld are both
present the magnetization rotates about the axis of the resulting effective ﬁeld.
FIGURE 13. Signals from 3 spins with different precession frequencies (left) and their corresponding Fourier
transforms (right)
I(t )
FT
 →  I(ν)
t
t
Α
Β
C
ν
Α Β
C
FT
I(det)
LECTURE COURSE: NMR SPECTROSCOPY
First Chapter: Physical Basis of the NMR Experiment Pg.15
This effective ﬁeld is calculated by taking the vector sum of the B
0
and B
1
ﬁeld:
Now, the B
0
ﬁeld is larger than the B
1
ﬁeld by many orders of magnitude (for
protons, the B
0
ﬁeld corresponds to precession frequencies of hundreds of
MHz whereas the B
1
ﬁeld is about 120 KHz). If the B
1
ﬁeld would be applied
ﬁxed along the xaxis all the time it would have an negligible inﬂuence onto
the spins. However, the B
1
ﬁeld is not static but rotates about the zaxis (the
axis of the static ﬁeld) with a frequency that is very similar to the precession
frequency of the spins about the zaxis. To understand the effect of the rotating
B
1
ﬁeld, it is very convenient to transform into a coordinate system that rotates
with the precession frequency ω
o
of the B
0
ﬁeld (in the picture above that
means that the small "blue" man jumps onto a platform that rotates with the
larmor frequency of the spin. This operation transforms from the laboratory
frame to a rotating frame, e.g. a frame in which the coordinate system is not
ﬁxed but rotates with ω
ο
):
For those spins whose lamor frequency is exactly ω
ο
the effective B
o
ﬁeld they
FIGURE 14. Movement of spins in the presence of only the B
0
ﬁeld (left), B
o
and B
1
ﬁeld (middle) and vector
additon to calculate the effective ﬁeld formed by B
o
and B
1
FIGURE 15. left: Static B
o
ﬁeld and rotating B
1
ﬁeld. middle: Laboratory frame frequencies, right: Rotating frame
frequencies.
B
o
B
1
B
eff
B
o
ω
0
B
o
ω
1
B
1
ω
0
ω − ω
0
ω B
o
B
1
ω
0
x
y
B
o
ω
B
o
ω
LECTURE COURSE: NMR SPECTROSCOPY
First Chapter: Physical Basis of the NMR Experiment Pg.16
experience in such a rotating reference frame is zero. These spins then only feel
the effect of the B
1
ﬁeld and precess about the B
1
axis as long as the B
1
ﬁeld is
turned on. Spins that have a slightly different precession frequency ω the
strength of the B
0
ﬁeld is
B’=B
0
ω/γ
(For ω=ω
0
, B’ is zero).
During RF pulses spins which are exactly onresonance (e.g. whose precession
frequency is equal to the precession frequency of the B
1
ﬁeld about the zaxis)
only feel the B
1
ﬁeld. These spins precess about the axis of the B
1
ﬁeld until the
B
1
ﬁeld is turned off again:
The overall ﬂipangle they have experienced during that time t
p
is
α = γ B
1
t
p
Usually the pulse lengths t
p
are choosen such that the ﬂip angle α is 90 or 180
degrees. As stated above the effect of the B
1
ﬁeld is to create phase coherence
amongst the spins. The macroscopic effect of coherent spins is transverse mag
netization.
1.6 Description of the effect of the B
1
ﬁeld on transverse and longitudinal
magnetization using the Bloch equations:
We have seen before that
Now,
B
x
= B
1
cosω
0
t
FIGURE 16. 1
st
: Vectorial addition of B
1
and B
o
ﬁeld. 2
nd
: Direction of the effective ﬁeld for spins exactly on
resonance, 3
rd
more offresonance, 4
th
far offresonance.
B’
B
1
B
eff
B
eff
B
eff
B
1
B’
B
0

ω
/
γ
B
1
B
eff
ω
o
ω
o
ω
o
t ∂
∂M
γ M B × ( ) =
LECTURE COURSE: NMR SPECTROSCOPY
First Chapter: Physical Basis of the NMR Experiment Pg.17
B
y
= B
1
sinω
0
t
B
z
= B
o
and hence
To solve these equations they are conveniently transformed into a reference
frame that rotates at the frequency ω
0
about the zaxis. Therein
In this "primed" coordinate system the equations become:
The solutions are rather involved and will not presented here since the signals
are usually recorded when only the B
0
ﬁeld is present nowadays. However, it
is important to see that in this frame the effect of the B
1
ﬁeld on transverse
magnetization aligned along the y' axis is to rotate it about x' with a frequency
of ω
1
= γ B
1
.
1.7 The excitation proﬁle of pulses:
A pulse is a polychromatic source of radiofrequency and it covers a broad band
of frequencies. The covered bandwidth is proportional to the inverse of the
pulse duration. Short pulses are required for uniform excitation of large band
widths, long (soft) pulses lead to selective excitation. Usually the nonselective
(hard) pulses have B
1
ﬁelds of the order of 520 kHz and pulselengths of 520
µs, whereas selective (soft) pulses may last for 1100 ms with an appropriately
t ∂
∂
M
x
t ( ) γ M (
y
B
0
M
z
B
1
ω
0
t ) sin + M
x
T
2
⁄ ( )
t ∂
∂
M
y
t ( ) γ M
x
B
0
M
z
– B
1
ω
0
cos t ( ) – M
y
T
2
⁄ ( )
t ∂
∂
M
z
t ( ) γ M
x
B
1
ω
0
t M
y
+ sin B
1
ω
0
cos t ( ) – M
z
M
0
– ( ) T
1
⁄ – =
– =
– =
M
x
′
M
x
ω
0
t M
y
ω
0
t
M
y
′
M
x
ω
0
t M
y
ω
0
cos t + sin =
sin – cos =
t ∂
∂M
x
′
ω
0
ω – ( )M
y
′
M
x
′
T
2
⁄ ( )
t ∂
∂M
y
′
ω
0
ω – ( ) – M
x
′
γ B
1
M
z
M
y
′
T
2
⁄
t ∂
∂M
z
′
–
γ B
1
M
y
′
M
z
M
0
– ( ) T
1
⁄ – – =
+ =
– =
LECTURE COURSE: NMR SPECTROSCOPY
First Chapter: Physical Basis of the NMR Experiment Pg.18
attenuated RF amplitude:
1.8 Relaxation:
The magnetization does not precess inﬁnitely in the transverse plane but
turnes back to the equilibrium state. This process is called relaxation. Two dif
ferent timeconstants describe this behaviour:
a) the reestablishment of the equiilibirum α/β state distri
bution (T1)
b) dephasing of the transverse component (destruction of
the coherent state, T2). The T2 constant characterizes the
exponential decay of the signal in the receiver coil:
The precessing spins slowly return to the zaxis. Instead of moving circularily
in the transverse plane they slowly follow a spiral trajectory until they have
FIGURE 17. Left: Excitation proﬁle of a “hard” pulse, right: Ex. proﬁle of a “soft” (long) pulse
FIGURE 18. Right: Signal in absence of transverse relaxation, right: real FID (free induction decay)
LECTURE COURSE: NMR SPECTROSCOPY
First Chapter: Physical Basis of the NMR Experiment Pg.19
reached their inital position aligned with the +/  zaxis:
A mechanical equivalent is a spring oscillating in time. The oscillation occurs
periodically so that the displacement from the equilibrium position follows a
cosine timedependence. Because of frictional energy loss the oscillation is
damped so that after some time the spring is not oscillating anymore:
The damping timeconstant is called T2 or transverse relaxation time. It charac
FIGURE 19. left: Trajectory of the magnetization, right: individual x,y,z component
FIGURE 20. Monitoring the position of a weight ﬁxed to a spring depending on the time elapsed after release can be
described by an damped oscillation.
LECTURE COURSE: NMR SPECTROSCOPY
First Chapter: Physical Basis of the NMR Experiment Pg.20
terizes the time it takes so that the signal has decayed to 1/ e of its original
magnitudeThe transverse relaxation constant T
2
is related to the linewidth of
the signals. The width of the signal at half height is given by:
Fast decay leads to broad signals, slow decay to sharper lines:
The transverse relaxation constant T
2
of spin I=1/ 2 nuclei is mainly governed
by
 the homogeneity of the magnetic ﬁeld ( the "shim")
 the strength of the dipolar interaction with other I=1/ 2 nuclei, depending
on the number and the distance of neighbouring nuclei
 the overall tumbling time of the molecule which is related to its size.
1.9 The intensity of the NMR signal:
If the α and β states would be populated equally, no netto change in energy
could be observed. The signal intensity is proportional to the population differ
ence of the two states.
The relative population of the states can be calculated from the Boltzmann dis
tribution:
with T being the measuring temperature and k the Boltzmann constant. E
β
E
α
FIGURE 21. Slowly decaying FIDs lead to narrow lines (left), rapidly decaying ones to broad lines (right).
∆ν
1 2 ⁄
1 πT
2
( ) ⁄ =
N
α
N
β
= e
(E
β
−E
α
)
kT
LECTURE COURSE: NMR SPECTROSCOPY
First Chapter: Physical Basis of the NMR Experiment Pg.21
is the energy difference between the α and the βstate.
Unfortunately, the energy difference in NMR experiments is very small, much
smaller than in IR or UV spectroscopy, and therefore the signal is much
weaker. In other words, the required quantities of sample are much larger. The
energy difference ∆E depends on the gyromagnetic ratio γ:
Since γ of
1
H is approx. 10 times larger for
1
H than for
15
N N
α
/ N
β
is much
larger and the signal much more intense. Furthermore, the natural abundance
of
1
H is 300 times larger than for
15
N. Direct observation of
15
N nuclei requires
high concentrations and long measuring times. ∆E also depends on B
o
. Higher
ﬁelds lead to dramatically reduced measurement times.
To be more precise the signal that is induced in the receiver coil depends on
both the gyromagnetic ratio of the excited and detected spin:
Int prop γ
ex
* γ
det
3/ 2
nucleus I
N
γ S
rel
Nat. Abd.
[10
8
T
1
s
1
] [%]
1
H 1/2 2.675 1.00 99.98
13
C 1/2 0.673 1.76 10
4
1.11
19
F 1/2 2.517 0.83 100
15
N 1/2 0.2712 3.85 10
6
0.37
31
P 1/2 1.083 0.0665 100
E = γ
h
2π
B
o
LECTURE COURSE: NMR SPECTROSCOPY
Second Chapter: Practical Aspects Pg.22
1. THE COMPONENTS OF A NMR INSTRUMENT
The NMR instrument consists mainly of the following parts:
• the magnet
• probehead(s)
• radiofrequency sources
• amplifiers
• analogdigital converters (ADC's)
• the locksystem
• the shim system
• a computer system
1.1 The magnet system:
Usually the static magnetic ﬁeld nowadays is provided by a superconducting (super
con) magnet. Therein, the main coil that produces the ﬁeld is placed in a liquid helium
bath so that the electric resistance of the coil wire is zero. The helium dewar is sur
rounded by a liquid nitrogen dewar to reduce loss of the expensive helium:
FIGURE 1. Schematic drawing of the magnet, showing He and N2 dewars, coils and probehead
liq. N
2
dewar
liq. He
dewar
Main Coil
Probehead
roomtemperature
shim tube
LECTURE COURSE: NMR SPECTROSCOPY
Second Chapter: Practical Aspects Pg.23
Additional socalled cryocoils are placed in the Hebath to correct partially for ﬁeld in
homogeneity. The magnet is vertically intersected by the roomtemperature shim tube.
On its surface the r.t. shim coils are located. These are the ones that have to be adjusted
by the user. The sample that is mostly contained in a deuterated solvent inside a 5mm
glastube is placed inside a spinner and then lowered through the r.t. shim tube so that it
enters the probehead from the top.
1.2 The probehead:
The probehead contains the receiver/transmitter coil (actually only a single coil for
both purposes):
However, most probes contain two coils: An inner coil tuned to deuterium (lock) and a
second frequency and an outer coil tuned to a third and possibly a fourth frequency.
The deuterium channel is placed on most probes on the inner coil. The inner coil is
more sensitive (higher S/N) and gives shorter pulselengths. An inverse carbon probe
has the inner coil tuned to
2
H and
1
H and the outer coil to
13
C. It has high proton sensi
FIGURE 2. View of the spinner with sample tube placed in probehead
˜
˜
˜
˜
Spinner
Resonance
Circuit
Cables to Preamp
Transmitter/
Receiver Coil
LECTURE COURSE: NMR SPECTROSCOPY
Second Chapter: Practical Aspects Pg.24
tivity and is wellsuited for inverse detection experiments like HSQC or HMBC. A car
bon dual probe has the inner coil tuned to
2
H and
13
C and the outer to
1
H and is
dedicated for carbon experiments with proton decoupling. The resonance circuit inside
the probe also contains the capacitors that have to be tuned when changing the sample
(especially when the solvent is changed). The receiver coil is in the center of the mag
netic ﬁeld. Once the spinner is lowered the NMR tube is positioned such that the liquid
inside is covered by the receiver coil completely. The receiver/transmitter coil should
not be confused with the cryocoils (coils placed in the He bath) that produce the static
ﬁeld. The probehead is then connected to the preampliﬁer which is usually placed close
to the magnet and which performs a ﬁrst ampliﬁcation step of the signal. The preampli
ﬁer also contains a wobbling unit required for tuning and matching the probe:
The radiofrequency sources (frequency synthesizers) are electronic components that
produce sine/cosine waves at the appropriate frequencies. Part of them are often modu
laters and phase shifters that change the shape and the phase of the signals. The fre
quency synthesizers nowadays are completely digital.Frequency resolution is usually
better than 0.1 Hz.
The ampliﬁers are lownoise audioampliﬁers which boost the outgoing and incoming
signals. They are in the range of 50100 Watts for protons and 250500 Watts for heter
onuclei.
The analogdigital converters (ADC's) are required because the signal is recorded
FIGURE 3. Magnet and preamplifier system
Preamp
Probehead
liq. N
2
Inlet
liq. He
System
LECTURE COURSE: NMR SPECTROSCOPY
Second Chapter: Practical Aspects Pg.25
in analog form (superposition of various damped harmonic oscillations) but
must be in digital form in order to be accessible to Fourier transformation by
the computer. In modern nmr spectrometers 16 or 18 bit digitizers are used.
This means that the most intensive signal can be digitized as 2
18
. This has
severe consequences for the dynamic range because it implies that signals with
less than 1/ 2
18
of the signal amplitude cannot be distinguished in intensity.
Therefore, the receiver gain (the ampliﬁcation of the signal) should be adjusted
such that the most intense signal almost completely ﬁlls the ampliﬁer. However,
some care has to be paid in experiments that utilize water suppression: Sometimes the
most intense signal in a 2D experiment does not occur in the ﬁrst increment because
the water may be (due to radiation damping) stronger in later increments. It is advis
able to set the evolution time to longer values (e.g. 100ms) and observe the intensity of
the water signal.
The computer system is an industrystandard UNIX machine with lots of RAM. How
ever, offside processing on PCs is also possible and the PC is also started being used
as host computers on the spectrometer (unfortunately).
1.3 The shim system:
Considering that the precession frequencies are proportional to the magnetic ﬁeld
strength the magnetic ﬁeld has to be highly homogenous across the sample volume in
order to be able to observe small frequency differences (small couplings). If the ﬁeld
would not be highly homogenous, the effective ﬁeld strengths in different volume com
partments inside the sample would be different and the spins therefore would precess at
different rates. This would lead to considerable linebroadening (inhomogeneous
broadening). The shimsystem is a device that corrects for locally slightly different
magnetic ﬁelds.
The shimsystem consists of two parts: (A) the cryoshimsystem and (B) the roomtem
perature shims. The basic principle behind them is the same. Small coils are supplied
with regulated currents. These currents produce small additional magnetic ﬁelds which
are used to correct the inhomogeneous ﬁeld created by the main coil. There are a num
ber of such coils of varying geometry producing correction ﬁelds in different orienta
tions. In the cryoshim system the coils are placed in the He bath, they are usually only
adjusted by engineers during the initial setup of the instrument. The roomtemperature
shim system is regulated by the user whenever a new sample is placed in the magnet or
when the temperature is changed.
The room temperature shim system is grouped into two sets of shims
• the onaxis (spinning) shims (z,z
2
,z
3
,z
4
..)
• the offaxis (nonspinning) shims (x,y,xy...)
LECTURE COURSE: NMR SPECTROSCOPY
Second Chapter: Practical Aspects Pg.26
The onaxis shims only correct for inhomogeneity along the zaxis. They are
highly dependent on the solvent and the solvent ﬁlling height. At least the
lower order gradients (z, z
2
and z
3
) should always be adjusted. The z
4
gradient
depends very much on the ﬁlling height and its adjustment is tedious. There
fore, in order to speed up the shimming process, the ﬁlling height should (if
possible) always be the same (e.g. 500 or 550 µl) and a shimset for that height
and probehead should be recalled from disk. The offaxis shims usually do not
have to be adjusted extensively (only x, y, xz and yz routineously). Wrong off
axis shims lead to spinning sidebands when the sample is rotated during mea
surement. Their contribution to the lineshape is largely removed with spinning
but this unfortunately introduces disturbances. It should therefore be avoided
during 2D and NOE measurements.
The name of the onaxis shims is derived from the order of the polynomial that needs to
be used to correct for the ﬁeld gradient along the zaxis:
The zshim delivers an additional ﬁeld that linearly varies along the sample
tube. The z
2
shim has its largest corrections to the ﬁeld at the top and the bot
tom of the sample.
Shimming is usually performed by either observing the intensity of the lock
FIGURE 4. Field dependence of onaxis shims
Bo
z
z2 z3
z4
For the more experienced spectroscopist the misadjusted onaxis shim can mostly be recognized from
the signal lineshape. Misadjusted shims with even exponentials (z
2
,z
4
,z
6
) give asymmetric signals,
those with odd exponentials (z
1
,z
3
,z
5
) show up as a symmetrical broadening. This is due to the symme
try of the function needed to correct of it. Z
4
for example increses the field strength at the the top and
the bottom of the sample and hence gives only frequencies larger than the correct frequency. Z
3
in con
trast increases the freuqnecy at the top and decreases the frequency at the bottom of the sample. There
fore, in the case of z4, there is a proportion of singal shifted to higher frequencies and hence to one side
of the signal, whereas z
3
give proportions of the signals shift to lower and higher frequencies resulting
in asymmetric and symmetric errors for the former and latter, respectively.
The closer the bump is to
the base of the signal the higher the gradients needs to be to correct for it.
LECTURE COURSE: NMR SPECTROSCOPY
Second Chapter: Practical Aspects Pg.27
signal (vide infra) or by monitoring the shape of the FID.:
Another way of controlling the homogeneity of the magnetic ﬁeld is to watch the shape
of the FID. When the ﬁeld is highly homogenous, the FID should fall smoothly follow
ing an exponential. The resolution of the signal determines how long the FID lasts:
FIGURE 5. Misadjusted shims and appearance of corresponding signals after FT.
FIGURE 6. FID of perfectly shimmed magnet (left) and misshimmed magnet (right).
LECTURE COURSE: NMR SPECTROSCOPY
Second Chapter: Practical Aspects Pg.28
1.4 The locksystem:
Stability of the magnetic ﬁeld is achieved by the deuterium lock system. The deuterium
lock measures the frequency of the deuterium line of the solvent. Hence, deuterated
solvents have to be used for FTNMR. The system has a feedback loop, which gener
ates corrections to the magnetic ﬁeld strength B
o
, such that the resonance frequency of
the solvent deuterium line remains constant. This is achieved by delivering a suitable
current to the z
0
shim coil. Consequently, all other resonance frequencies are also kept
constant. Usually the lock system has to be activated when the sample has been placed
in the magnet. When the locksystem is not activated the naturally occurring drift of the
magnetic ﬁeld leads to varying resonance frequencies over time and hence to line
broadening.
The stability of the lock system is critical for many experiments, mostly 2D measure
ments and even more importantly, NOE measurements. The stability is inﬂuenced by
many factors of which the temperature instability has the largest inﬂuence. To provide
good sensitivity of the lock the lock power should be adjusted just below saturation
(lockline must still be stable!).
Modern digital lock system allow to adjust the regulation parameters of the lock chan
nel.Thereby, one can determine how fast the lock circuit reacts (the damping of the
lock). For experiments utilizing pulsedﬁled gradients the lock should be highly
damped in order to avoid that the lock tries to correct each time the ﬁeld recovers from
the gradient!
1.5 The transmitter/receiver system:
It is much more convenient to handle audiofrequencies than handling radiofrequen
cies. We have seen before that the B
1
ﬁeld rotates about the axis of the static ﬁeld with
a certain frequency, the carrier frequency, that coincides with the center frequency of
the recorded spectrum. Similarly, the received signal is also transformed down to audio
frequency. In addition we require quadrature detection: A single coil cannot distin
guish positive and negative frequencies, that means distinguish frequencies of spins
rotating slower than the carrier frequency from those that precess at higher rates:
FIGURE 7. Top: Signal due to negative frequency. Bottom: Signal due to positive frequency,
0 +ν  ν
a) single detector
0 +ν  ν
LECTURE COURSE: NMR SPECTROSCOPY
Second Chapter: Practical Aspects Pg.29
In principle, such a separation could be achieved by using two receiver coils which dif
fer by 90° in phase (a quadrature detector):
One could would then detect the cosine modulated and the other the sinemodulated
component of the signal. Adding sine and cosine modulated components of the signal
cancels the “wrong” frequency component:
However, probeheads contain only one coil. Sineand cosinemodulated components of
the signal result from a different trick: The signal that comes from the receiver coil
(which of course is HF (MHz)) is split into two. Both parts are mixed with the transmit
ter (carrier) frequency [for Bruker machines: SFO1], the frequency with which the B
1
ﬁeld rotates. However, the phase of the transmitter frequency that is added differs by 90
degrees for the two parts. Thereby, the radiofrequencies (MHz range) are transformed
into audiofrequencies (kHz range). These can be handled in the following electronics
much easier. We do see now, that both the transmitter and the receiver system effec
tively work in the rotating frame. The rotating frame was introduced in the transmitter
system by having the B
1
ﬁeld rotating in the x,y plane and in the receiver system by
subtracting the transmitter frequency from the signal. What we actually measure are
therefore not MHzfrequencies but frequencyoffsets (differences to) the carrier fre
quency. In addition, we have introduced quadrature detection:
Transmitting and receiving is done on the same coil. The signal coming from
FIGURE 8. Sign discrimination from quad. detection
FIGURE 9. Fourier transform of a sin (top) and cosine wave (bottom).
b) quadrature detector
0 +ν  ν
0 +ν
 ν
sin ωt
cos ωt
Σ
FT
FT
LECTURE COURSE: NMR SPECTROSCOPY
Second Chapter: Practical Aspects Pg.30
the coil is blanked for a short time after the pulses (the socalled prescan
delay) and then used for detection. The transmitter/ receiver system contains
mainly analog parts.
When cosine and sinemodulated signals are slightly differently ampliﬁed socalled
quadrature images remain. They are usually observed for intense signals when few
scans are recorded and can be easily recognized because they are mirrored about the
FIGURE 10. Scheme of the RF path for a 300 MHz NMR spectrometer (proton detection).
Splitter
90° paseshifter
sin ωt
cos ωt
Mixer
SFO1
(300.13 MHz)
Amplifier
FT
+
HF
(MHz)
Audio
( 0KHz)
AnalogDigital Converter
ADC
LECTURE COURSE: NMR SPECTROSCOPY
Second Chapter: Practical Aspects Pg.31
zerofrequency (the middle of the spectrum). Therefore, modern instruments utilize
“oversampling”. A much larger spectral width is sampled, such that the quadrature
images do not fall into the observed spectrum any longer. Prior to processing the non
interesting part of the spectrum is removed.
2. BASIC DATA ACQUISITION PARAMETER
The following paragraph summarizes the parameters that govern the acquisition of 1D
spectra and hence have to be properly adjusted before the measurement.
The 1D spectrum is characterized by the frequency in the center of the spectrum and by
its width. Remember that the B
1
ﬁeld rotates about the axis of the static ﬁeld with a cer
tain frequency:
Exactly this frequency is mixed with the signal that comes from the receiver coil and
therefore is effectively subtracted from the signal frequency. Hence, the signal is mea
sured in the rotating frame and the frequencies will be audio frequencies (0 kHz). If
the precession frequency of a particular spin is exactly the same as the frequency with
which the B
1
ﬁeld rotates it will have zero frequency and appear in the center of the
spectrum. The frequency of the rotating B
1
ﬁeld is called the (transmitter) carrier fre
quency:
sfo1: spectrometer frequency offset of the ﬁrst (observe) channel
this parameter determines the carrier offset. Frequencies larger than sfo1 will be on the
left side of the spectrum, smaller frequencies on the right side. sfo1 is the absolute fre
quency (in MHz). If you want to make a small change of the carrier position, you can
change the offset o1
FIGURE 11.
FIGURE 12. Basic parameter for specifying the acquired spectral region
ω
0
ω
B
o
ω
1
ω
B
o
B
1
ω
0
x
y
spectral width (sw)
transmitter frequency
(sfo1)
0  sw/ 2 + sw/ 2
LECTURE COURSE: NMR SPECTROSCOPY
Second Chapter: Practical Aspects Pg.32
sfo1 = bf1 + o1
bf1 is the socalled basicspectrometer frequency, e.g. 500.13 MHz and o1 is the offset,
a frequency in the audio (0> KHz) range.
sfo2: spectrometer frequency offset of the second (decoupler) channel
like sfo1, required for example for acquisition of proton decoupled carbon spectra. The
proton frequency is then deﬁned on the second channel and should correspond to the
center of the proton spectrum.
Now, the next question is, how to adjust the spectral width (the width of the spectrum
in Hz). This done by proper choice of the dwell time. The signal is sampled stroboscop
ically and neighboring data points are separated by the dwell time. The Nyquist theo
rem says that at least one point per period must be sampled in order to characterize the
frequency correctly. Since the highest frequency is sw (in Hz), the dwell time must be
dw = 1/sw
Signals that have a higher frequency have made more than a 360 degree rotation
between the sampling points. On older instruments (AMX spectrometers) they are
attenuated by the audioﬁlters. They are recognized from the fact that they cannot be
phased properly, usually. On the modern instruments (those with oversampling, DRX
series), signals outside the spectral width completely disappear. Therefore, it is always
recommended to record the spectrum over a very large spectral width initially to make
sure that no signals at unusual places (e.g. OH protons at 20 ppm or signals below 0
ppm) are lost.
FIGURE 13. Left: Sampled data points. Right: Black: Sampled data points. Red: Missing sampling point for a
frequency of twice the nyquist value..
AQ
DW
(td =16)
LECTURE COURSE: NMR SPECTROSCOPY
Second Chapter: Practical Aspects Pg.33
sw: spectral width
gives the width of the acquired spectrum (on Bruker instruments, sw is in units of ppm,
swh in units of Hz).
dw: dwell time
is the time spacing between sampling of two consecutive data points.
dw = 1/sw or dw = 1/2sw
The dwell time is determined by the inverse of the spectral width (or the inverse of
twice the spectral width, depending on the quadrature detection mode).
td: time domain data points
The number of points that are sampled is called "time domain" data points (td). The
longer the FID is sampled the better will be the resolution in the spectrum provided
there is still signal in the FID.
aq: acquisition time
determines the total time during which a single FID is sampled
aq = td * dw
for 1D spectra, aq is set such that the signals have completely decayed towards the end
of the FID (which corresponds to the transverse relaxation time T2). If aq is set to a
shorter value, the resolution is lower, if aq is >> T2, only noise will be sampled at the
end of the FID, and therefore the experimental time is unnecessarily prolonged.
The complete measuring time for a simple 1D spectrum is approx.
t
tot
= ns (RD+aq)
in which ns is the number of scans and RD the relaxation delay (vide infra).
ns: number of scans
the number of FIDs which are coadded. The signal to noise ratio of a measurement
increases as
which means that in order to improve the S/N by a factor of two the measurement time
must be increased by a factor of 4. Sometimes, the minimum number of ns is deter
mined by the phasecycle of an experiment.
RD: relaxation delay
time between end of sampling of the FID and start of the next experiment. On Bruker
instruments, the relaxation delay is characterized by the parameter d1. The effective
time for relaxation is given by the repetition rate
S
N
∝ ns
LECTURE COURSE: NMR SPECTROSCOPY
Second Chapter: Practical Aspects Pg.34
t
r
= aq + d1
rg: receiver gain
This is the ampliﬁcation of the signal. Settings are possible in powers of 2 (e.g.
1,..,128,256,...32k). For automatic adjustment of the receiver gain on spectrometers use
rga. The receiver gain has to be set such that the maximum intensity of the FID ﬁlls the
ADC (analogdigital converter). If the receiver setting is too low, the dynamic range of
the instrument is not exploited fully and very small signals may be lost. If the receiver
gain setting is too high, the ﬁrst points of the FID are clipped, which will lead to severe
baseline distortions.
p1,p2,..length of pulses [in µs]
length of pulses in µs. This is the duration that
the B
1
ﬁeld is switched on. The B
1
ﬁeld rotates
about the zaxis with the frequency ω
o
. We
have seen before that if the frequency ω with
which the spin precesses about the zaxis is
identical to ω
o
it does not feel B
0
in the rotat
ing frame.It will then precess about the axis of
the effective ﬁeld which in that case corre
sponds to the axis of the B
1
ﬁeld. However, the
precession frequency about the B
1
axis, ω
1
, is much smaller, it is in the kHz
range (or smaller). The strength of the B
1
ﬁeld is correlated to the time required
for a 360 degree rotation PW
360
via
ω
1
=
γB
1
/ 2π = 1/ (PW
360
)
Usually either 90 or 180 degree rotations are required in NMR. The following conven
tions are made in pulseprograms:
• p190˚ transmitter pulse length
• p2180˚ transmitter pulse length
• p390˚ decoupler pulse length
• p4180˚ decoupler pulse length
FIGURE 14. Definition of the recycle delay.
AQ D1
tr
ω
o=SFO1
ω
B
o
ω
1=(4*(PW90)
)
1
LECTURE COURSE: NMR SPECTROSCOPY
Second Chapter: Practical Aspects Pg.35
The ﬂip angle of the pulse Φ is determined not only by the length of the pulse
τ
p
, but also by the power level, at which the B
1
ﬁeld is applied:
Φ = τ
p
γB
1
For proton pulses, the power level is given by the parameters hl1,hl2,hl3,..hl6 (hl=
1
H
level). For pulses on the heteronuclei (
13
C,
15
N,
31
P ...), power setting parameters are
tl0,tl1,..tl6 (transmitter level 1,2..) or dl0,dl1..dl6 (decoupler
level 1,2..) for the low power mode. In the default mode, heteronuclear pulsing is done
in the highpower mode and cannot be regulated (always full power). On the newer
DRX instruments the power levels are speciﬁed as pl1:f1 (power level 1 on channel 1).
LECTURE COURSE: NMR SPECTROSCOPY
Third Chapter: Practical Aspects II Pg.36
1. ACQUISITION OF 1D SPECTRA
Acquisition of simple 1D spectra comprises the following steps
• changing the probehead
• Setting the correct temperature for measurement
• Correct positioning of the tube in the spinner and insertion of the sample into
the magnet
• recalling a shimset with approximately correct shims for the solvent and
probehead
• activation of the field/frequency lock system
• adjustment of the homogeneity of the magnetic field (shimming)
• recalling a parameter set for the desired experiment
• (tuning of the probehead)
• (calibration of the pulse lengths)
• determination of the correct receiver gain setting
• performing a preliminary scan with large spectral width (
1
H only, approx. 20
ppm)
• acquisition of the final spectrum with sufficient S/N and optimized spectral
width
1.1 Calibration of pulse lengths:
The ﬂipangle θ of pulses is proportional to the duration (τ
p
) and strength of
the applied B
1
ﬁeld:
θ = γB
1
τ
p
It is also obvious that for nuclei with lower γ, higher power setting must be
applied (typically for
1
H: 50 W, for
15
N:300W). The length of the pulses has
therefore to be determined for a given power setting. For calibration of proton
pulses, a spectrum is acquired with setting the length of the pulse correspond
ing to a small ﬂip angle, phasing of the spectrum, and then increasing the
pulselength in small steps. When the length of the pulse is set corresponding
to a 180˚ pulse, a zerocrossing of the signal is observed, as well as for the 360˚
pulse. The length of the 90˚ pulse can be calculated to be half of the length of
LECTURE COURSE: NMR SPECTROSCOPY
Third Chapter: Practical Aspects II Pg.37
the 180˚ pulse:
13
C pulses are most easily determined on a
13
Clabeled sample such as an ace
tate sample (labeled at the Me carbon). Calibration is performed in the so
called inversedetection mode with the following sequence detecting the pro
ton signal (which is split by the
13
C coupling into an antiphase doublet):
When the carbon pulse is exactly set to 90˚, all magnetization is converted into
multiplequantum coherences, and no signal is observed:
FIGURE 1. Calibration of pulse length. The length of the proton pulse is incremented by a fixed amount for each
new spectrum.
FIGURE 2. Pulse sequence to determine the 90° carbon pulse with proton detection
FIGURE 3. Resulting spectra from the sequence of Fig.2.
1
H
13
C
1/2J(C,H)
1us
5us
10 us (90 deg.)
15us
J(C,H)
LECTURE COURSE: NMR SPECTROSCOPY
Third Chapter: Practical Aspects II Pg.38
1.2 Tuning the probehead:
The purpose of the radiofrequency circuit inside the probe is to deliver a rotat
ing B
1
ﬁeld and to detect the signal. An oscillating electromagnetic ﬁeld is also
part of a radio and hence the resonance circuit inside the probe has much in
common with the one found in a radio:
A resonance circuit has two basic components: a capacitor (C) and a coil (or
inductance, L). The energy is interchangingly stored in form of an electric or
magnetic ﬁeld in the capacitor or in the coil, respectively. The frequency of
oscillation is
in which L is the inductance of the coil and C the capacity.
In order to get best sensitivity and the shortest pulselengths the probe has to be
• tuned to the transmitter frequency
• the impedance should be matched to 50 Ω.
When properly tuned and matched, the probe has optimum sensitivity and the
13
C pulse lengths for example do not have to be measured on the sample but
predetermined values can be used instead. A large inﬂuence to the tuning and
matching is caused by the dielectric constant of the sample. The reason for this
is, that if the sample is introduced into the inner of the coil the dielectric con
stant in that part and hence the impedance of the coil changes. Care has be
taken when solvents with high salt contents are used: Even in the case of per
fect matching and tuning the pulselengths can be considerably longer (about
1.5 times for protons when using 150mM NaCl), although this effect largely
depends on the nucleus and is most pronounced for proton frequencies. Tun
ing and matching can be performed in different ways: (A) the reﬂected power
FIGURE 4. Basic principle of the probehead circuit.
L
C
E
B
ω 1 LC ( ) ⁄ =
LECTURE COURSE: NMR SPECTROSCOPY
Third Chapter: Practical Aspects II Pg.39
of the probe during pulsing can be minimized. (B) Nowadays wobbling is used
during which the frequency is swept continuously through resonance covering
a bandwidth of 420 MHz:
Modern probes usually have two coils, an inner coil and a outer coil, and the
inner one has higher sensitivity. An inverse probe, which is optimized for
1
H
detection has
1
H frequency on the inner and
13
C frequency on the outer coil.
The dual probe has higher sensitivity for
13
C, and consequently, the inner coil
is used for 13C:
Recently, socalled cryoprobes have been introduced. For these probes the RF
coil and the preampliﬁer is at Hetemperature. Thereby, the thermal noise level
is largely reduced (the amount of signal is even a little less than for a conven
tional probe) and the signal/ noise in the absence of salt increased by almost a
factor of four.
1.3 Adjusting the bandwidth of the recorded spectrum:
The bandwidth of the spectrum is determined by the dwell time (dw), the time
spacing between recording of two consecutive data points (vide supra).
FIGURE 5. Wobbling curve of a detuned probe (thick line). By tuning, the minimum is moved along the horizontal
axis and by matching the minimum becomes deeper. The optimum setting is shown as a dotted line.
FIGURE 6. Arrangement of coils for inverse and direct carbon detection probeheads
SFO1
tuning
m
a
t
c
h
i
n
g
4
9
0
5
1
0
≈ ≈
Inverse
Dual
1
H
1
H
13
C
13
C
LECTURE COURSE: NMR SPECTROSCOPY
Third Chapter: Practical Aspects II Pg.40
If the sampling rate is too low, the sig
nal will still be sampled but will have a
wrong frequency after Fourier transfor
mation. Although analog or digital ﬁl
ter decrease the intensity of folded
peaks, they may well be in the spec
trum. On the other hand, signals may
easily become lost, if the spectral width
is set too small. The best way to prevent folding or loosing of signals is to
record a preliminary experiment with a very large bandwidth (e.g. 30 ppm for
1
H) and then adjusting offset and spectral width to cover all signals. Usually
10% are added to the edges because the audio ﬁlters tend to decrease signal
intensity of signals close to the edge. Folded peaks can be recognized, because
usually they are not inphase after adjustment of zero and ﬁrst order phase cor
rection. Alternatively, they change their position in the spectrum, when the off
set is varied slightly.
However, care has to be taken on the newer instruments that use oversampling and dig
ital filters. In this case the frequencies outside the sampled region will be almost com
pletely removed by the digital filters!
1.4 Data processing:
Once the analog signal that comes from the coil has passed the ampliﬁers, it is
converted into a number by the analogtodigital converter (ADC). The result
ing numbers which represent signal intensity vs. time [f(t)] are then converted
into a spectrum that represents signal intensity vs. frequency [f(ω)] by use of
the Fourier Transformation.
The Fourier theorem states that every periodic function may be decomposed
into a series of sine and cosine functions of different frequencies:
The Fourier decomposition therefore tells us which frequencies are present to
FIGURE 7. Effect of folding (aliasing) of signals.
f x ( ) a
0
2 ⁄ a
n
nx b
n
nx sin
n 1 =
∞
∑
+ cos
n 1 =
∞
∑
+ =
LECTURE COURSE: NMR SPECTROSCOPY
Third Chapter: Practical Aspects II Pg.41
which amount (with which intensity). Using the integral form of the equation
above
and utilizing the Euler relation the equation can be rewritten as:
This is the recipe to transform the signal function F(t) which for a single reso
nance is
into the spectrum F(ω). Analogously, the FT can be used to transform from the
frequency into the timedomain:
Note that this transformation is the continuous FT (used for continuous func
tions). Usually, the integral is split up again into the cosine and sine terms cor
responding to the cosine and sine transforms, so that
Note that in FTNMR the signal is sampled as discrete points. Hence, the dis
crete FT has to be utilized, which transforms a Npoint time series consisting of
values d
k
into a Npoint spectrum f:
The discrete FT is implemented in form of the very fast CooleyTukey algo
rithm. The consequence of using this algorithm is, that the number of points the spec
trum has must be a power of 2 (2
n
).
f ω ( ) 1 2π ( ) ⁄ a t ( ) ωt ( ) b t ( ) ωt ( ) sin + cos t
a t ( )
d
∞ –
∞
∫
1 2π ( ) ⁄ f x ( ) tx ( ) cos x
b t ( )
d
∞ –
∞
∫
1 2π ( ) ⁄ f x ( ) tx ( ) sin x d
∞ –
∞
∫
=
=
=
f ω ( ) 1 2π F t ( )e
iωt –
t d
∞ –
∞
∫
⁄ =
F t ( ) e
iωt –
e
t T2 ⁄ –
• =
F t ( ) 1 2π ( ) ⁄ f ω ( )e
iωt –
ω d
∞ –
∞
∫
=
f t ( )e
iωt
t d
∞ –
∞
∫
Re f ω ( ) [ ] Im f ω ( ) [ ] +
f t ( ) ωt cos t i f t ( ) ωt sin t d
∞ –
∞
∫
+ d
∞ –
∞
∫
=
=
f
n
1 N ( ) ⁄ d
k
e
2πikn N ⁄ –
k 0 =
N 1 –
∑
=
LECTURE COURSE: NMR SPECTROSCOPY
Third Chapter: Practical Aspects II Pg.42
The next ﬁgure will summarize the FT of some important functions (the so
called Fourier pairs):
The FT of a cosinewave gives two deltafunctions at the appropriate frequency
(A). The FT of a decaying exponential gives a lorentzian function with a char
acteristic shape (B). The FT of a properly shimmed sample containing a single
frequency gives two signals at the appropriate frequency with lorentzian line
shape (C). In fact the FT of (C) can be thought of a convolution of (A) and (B).
The FT of a step function gives a sinc function
The sinc function has characteristic wiggles outside the central frequency band
(D). A FID that has not decayed properly can be thought of as a convolution of
a step function with an exponential which is again convoluted with a cosine
function. The FT gives a signal at the appropriate frequency but containing the
wiggles arising from the sinc function (E). A gaussian function yields a gauss
ian function after FT. Signals that have not decayed to zero are common in 2D
FIGURE 8. Results of different functions after Fourier transformation in frequency space.
FT
A
B
C
D
E
F
c x ( ) sin x ( ) sin x ⁄ =
LECTURE COURSE: NMR SPECTROSCOPY
Third Chapter: Practical Aspects II Pg.43
NMR were the number of data points sampled is restricted. In these cases suit
able window functions are used to force the signal to become zero towards the
end of the FID.
1.5 Phase Correction
We have seen that the complex Fourier transform can be decomposed into a
cosine transform and a sine transform that yield the real and imaginary parts
of the signals, respectively. In practice, real and imaginary parts of the signal
are stored in separate memory locations. However, the real part that is used for
displaying the spectrum does not necessarily need to be purely absorptive:
Purely absorptive signals have a much narrower base, so that differentiation of
peaks very close to each other is easier if they have been phased to yield purely
absorptive line shape. Therefore, a phasecorrection φ is needed to yield purely
absorptive signals. It will do the following manipulations:
with Re(∆φ=0) and Re(∆φ) being the real part of each data point before and
after the phase correction, respectively.
The phase of the signals after Fourier Transformation depends on the ﬁrst
point of the FID. If the oscillation of the signal of interest is purely cosinemod
ulated, the signal will have pure absorptive line shape. If the signal starts as a
pure sinemodulated oscillation, the line shape will be purely dispersive:
FIGURE 9. Signal in pure absorption or dispersion mode
1/ πT
2
absorption
mode
dispersion
mode
Re ∆Φ ( ) Im ∆Φ 0 = ( ) φ Re ∆Φ 0 = ( ) + Φ
Im ∆Φ ( ) Re ∆Φ 0 = ( ) φ Im ∆Φ 0 = ( ) + Φ cos sin =
cos sin – =
LECTURE COURSE: NMR SPECTROSCOPY
Third Chapter: Practical Aspects II Pg.44
There are two different types of phasecorrection. The zeroorder phase correction
applies the phase change to all signals in the same way. The ﬁrst order phase cor
rection applies a phase change, whose amount increases linearly with the dis
tance to the reference signal (marked with an arrow in the ﬁgure below):
What is the origin of the varying phase in the spectra? The zeroorder arises
because the absolute phase of the signals at the detector depends on the cable
lengths etc. The linear dependence of the phase of the signals has a more com
plicated reason. In a theoretical 1D experiment, acquisition of the FID would
start immediately after application of the pulse. However, in a real experiment
there is a protection delay (called de on Bruker instruments), to wait for the
pulse ringdown (pulsing and detection of the signal is done on the same coil,
so that there must be a protection delay after the pulse):
FIGURE 10. The relative phase of the magnetization with respect to the receiver coil determines the phase of the
signal after Fourier transformation
FIGURE 11. Top: The zero order phase correction changes the signal phase for all signals by the same amount.
Lower: For the first order phase correction the applied correction depends on the frequency difference to the
reference signal (marked by an arrow).
FIGURE 12. A) Ideal FID. B) Introduction of the prescan delay to enable pulse power ringdown.
t = 0
t = 0
PC
"0
th
order"
PC
"1
st
order"
DE
A
B
LECTURE COURSE: NMR SPECTROSCOPY
Third Chapter: Practical Aspects II Pg.45
During the protection delay de, the spins start to precess. The precession fre
quency depends on the offset of the signal frequency, e.g. the distance of the
signal from the middle of the spectrum. The ﬁrstorder phase correction
reverses the phase change due to shift evolution during de and is therefore lin
early dependent on the frequency of the signal:
To adjust the phase, go into the phase correction mode, deﬁne a signal at one
end of the spectrum as the reference phase, use the zeroorder phase correction
to phase it to absorption and then use the ﬁrst order phasecorrection to phase
the signal at the other edge of the spectrum. All other but folded signals will then
be in phase.
When spectra cannot be phased a magnitude calculation or the power spectrum
(PS=M
2
) is used:
FIGURE 13. Precession of spins with different frequencies during the prescan delay.
FIGURE 14. (A) normal FT with phase correction.(B) Power spectrum. (C) Magnitude calculation.
A
B
≈
≈
B
LECTURE COURSE: NMR SPECTROSCOPY
Third Chapter: Practical Aspects II Pg.46
The magnitude calculation yields much broader peaks at the base. Both, mag
nitude and power calculation lead to a severe loss in resolution.
1.6 Zeroﬁlling and the resolution of the spectrum
The resolution that can be obtained from a spectrum is determined by two fac
tors:
• the natural line width that is related to the transverse relaxation time
(homogenous broadening) and which is due to efficient dipolar coupling of the
spins. Fast transverse relaxation may also be caused by field inhomogeneity
(inhomogenous broadening).
• the number of points which are used to digitize the signal. If the number of
points after FT (on Bruker instruments the parameter SI) is smaller than the
number of acquired data points (on Bruker instruments the parameter TD), the
signal can not be digitized properly, and the resolution is very low:
The theoretical resolution obtainable from the FID can be calculated as
Res = SW (Hz) / TD
neglecting relaxation effects.
Provided that the FID has decayed to zero towards its end it is useful to
FIGURE 15. Left:Effect of increasing number of data points after FT. Right: Spectrum without zerofilling (lower
trace) and after zero filling with 5*TD (upper trace).
M Re
2
Im
2
+ =
LECTURE COURSE: NMR SPECTROSCOPY
Third Chapter: Practical Aspects II Pg.47
append zeros to the FID. Adding zeros once (that means making si = 2*td)
gives a signiﬁcant improvement in resolution, adding more zeros gives only
cosmetic effects but does not add more information.
When the FID has not decayed to zero, adding zeros introduces a step function
that causes sinctype artifacts (wiggles).
1.7 Resolution enhancement and S/N improvement
The resolution is determined by the transverse relaxation time T2 of the spins.
A long transverse relaxation time leads to a slowly decaying, fast relaxation to
a quickly decaying FID. Therefore, the resolution is determined by the amount
of signal which remains towards the end of the FID. Any data manipulation,
e.g. multiplying the FID with a increasing exponential (e
t/ x
) will improve the
resolution. Conversely, the S/ N is determined by the amount of signal at the
beginning of the FID. Most manipulations that improve resolution lead to a
loss in S/ N.
In
13
C nmr, S/ N is comparably bad, whereas resolution is rarely a problem.
Data processing of
13
C spectra therefore usually is performed with exponential
multiplication with a decaying exponential (em with LB>=1). In
1
H nmr, S/ N
is rarely a problem, but good resolution is required, and data could be multi
plied with an increasing exponential (em with LB< 0).
Application of the window function amounts to multiplying the FID data
point d
k
by
d
k
’=d
k
*a
k
in which k runs from 0 to TD (the total number of data points).
There are number of socalled window functions that are aimed at either
improving resolution or S/ N and their effect on a spectrum is shown in the ﬁg
ure below:
FIGURE 16. Top: Resulting FT of a FID that did not decay to zero in comparison to a fast decaying FID (bottom)
LECTURE COURSE: NMR SPECTROSCOPY
Third Chapter: Practical Aspects II Pg.48
Important, frequently used window functions are
1.7.1 Exponential multiplication:
The line broadening constant lb can be positive (sensitivity enhancement) or
negative (resolution enhancement). A lorentzian line of width W will have a
width of W + LB Hz after apodization. It is mainly used for processing
13
C
data, for which sensitivity enhancement is desired but where resolution is
rarely a problem (use lb = 12 Hz).
1.7.2 LorentztoGauss transformation:
This a combination of exponential and gaussian multiplication.
The factor lb determines the broadening, whereas gb determines the center of
the maximum of the gaussian curve. When gb is set 0.33, the maximum of the
FIGURE 17. (A) raw spectrum after FT. (B)Multiplication with exponential,LB=5.(C) as (B), but LB=1. (D) sine
bell.(E) 45 degree shifted sinebell.(F)90 degree shifted sinebell.
a
k
e
πlbk∆t –
=
a
k
e
πlbk∆t
e
gb k∆t ( )
2
–
• =
LECTURE COURSE: NMR SPECTROSCOPY
Third Chapter: Practical Aspects II Pg.49
function occurs after 1/ 3 of the acquisition time.
By this multiplication, the Lorentzian lineshape is converted into a gaussian
lineshape, which has a narrower base:
1.7.3 SineBell apodization:
Sinebell apodization is frequently used in 2D NMR processing. A pure sine
bell corresponds to the ﬁrst halflobe of a sinewave and multiplies the FID
such that it brought to zero towards the end. However, such a function also
sets the ﬁrst data points to zero and hence leads to severe loss of S/ N and
introduces negative wiggles at the bases of the signals in addition to a strong
improvement in resolution. Shifting the sinebell by 90 degrees (a cosinebell)
means that the function used starts of the maximum of the ﬁrst lobe and
extends to zero towards the end of the FID. This is usually used for FIDs that
have maximum signal in the ﬁrst data points (NOESY, HSQC). A pure sinebell
is used for such experiment in which the FID does not have maximum signal in
the ﬁrst points (such as COSY) or for improving magnitude data (HMBC):
FIGURE 18. Left: Lorentzian lineshape. Right: Gaussian lineshape.
FIGURE 19. Left: FID of COSY with shifted sinebell.Right: FID of NOESY with cosinebell
LECTURE COURSE: NMR SPECTROSCOPY
Third Chapter: Practical Aspects II Pg.50
The inﬂuence of the parameters on the window functions is shown below:
An often encountered problem is truncation of the signal, which means, that
the signal is not sampled until it has completely decayed. This is the case,
when the data matrices, e.g. the number of data points, must be kept to a rea
sonable low value, e.g. in 2D NMR, or when one very slowly decaying line is
present in the spectrum. Then the signal can be described by a superposition of
an oscillation, an exponential and a square function. The contribution from the
square function will introduce wiggles at the base of the signals which are orig
inating from the sinc function. Such a problem can be circumvented if the FID
is multiplied with a function that decays to zero at the end of the FID, e.g. an
decaying exponential or a cosine bell function.
1.8 Baseline Correction
Baselines that are not ﬂat are a major problem in FTNMR. Firstly, spectra with
nonﬂat baselines give wrong integrals. Secondly, in spectra with baseline roll
small signals may not be recognized. Thirdly, those spectra are difﬁcult to
phase. Furthermore, peakpicking routines need a threshold for minimal signal
intensity and such peakpicking for tiny signals is not possible when the base
line is not ﬂat. There are many reasons for bad baselines. In general, baseline
distortions are caused by distortions of the ﬁrst points of the FID. A frequent
source is receiver overload, that means, that the receiver gain has been set too
high. The ADC (analogtodigital converter) can only digitize a certain maxi
mal signal intensity. On the Bruker instruments, that means that the FID must
ﬁt into the acquisition window completely. If the signal intensity is larger, the
ﬁrst points of the FID are clipped:
FIGURE 20. Different window functions. A: exponential multiplication with LB=1,3,5Hz. B) LorentzGaussian
transformation with LB=5 and GB =0.1, 0.3, 0.5 Hz. C) Sinebell shifted by 0, π/2, π/4, π/8. D) Sinebell squared
shifted by 0, π/2, π/4, π/8.
LECTURE COURSE: NMR SPECTROSCOPY
Third Chapter: Practical Aspects II Pg.51
Such a baseline cannot be corrected, and the measurement must be repeated
with a lower receiver gain setting. Other baseline problems, like baselinedrift
(nonhorizontal but ﬂat baseline) or baselinecurvature can be corrected, on
Bruker machines either automatically (with the command abs) or manually.
Oversampling has shown to reduce baseline problems in the directly sampled
frequency dimensions dramatically.
Frequently, one can recognize which data point is distorted from the shape of
the baseline. A wrong ﬁrst data point leads to an offset, a wrong second one to
a halfwave, a wrong third one to a full wave, etc. In principle, spectra with (a
few) wrong ﬁrst data points can still be used, if linear backprediction of the ﬁrst
data points is used.
1.9 Linear prediction:
For exponential decaying signals that are sampled at constant spacing (as is
usually the case for NMR) new data points can be predicted. The reason
behind this is that one can assume that new data points can be represented as a
ﬁxed linear combination of immediately preceding values. LP provides a
means of ﬁtting a time series to a number of decaying sinusoids. Provided that
S/ N is good the number of points can be doubled with LP (or the measuring
time can be halved to achieve the same resolution). Predicting more points
severely changes the lineshape of the signals and is not recommended. LP
gives very good results for COSY and HSQC data. Especially in the case of the
FIGURE 21. lower trace: spectrum after FT of an FID with correctly adjusted receiver gain. Top: Spectrum after FT
of a clipped FID.
LECTURE COURSE: NMR SPECTROSCOPY
Third Chapter: Practical Aspects II Pg.52
COSY, antiphase signals cancel when the resolution is low. Linear prediction in
these cases not only increases the resolution but also increases the amount of
signal!
With modern computing power it is performed rather quickly and the results
can be astonishing good. Remember that in order to double the resolution in
the indirect dimension the number of experiments in a 2D spectrum and hence
the acquisition time has to be doubled. Linear prediction may achieve the same
goal in a few seconds. In the case of constanttime experiments, mirrorimage
linear prediction may be applied which is capable to predict N points to 4*N
points.
FIGURE 22. ctHSQC spectra (A) 240 points.(B) 64 points.(C) LP to 128 points.(D) mirror image LP to 240
points.
LECTURE COURSE: NMR SPECTROSCOPY
Fourth Chapter: Chemical shifts and scalar couplings Pg.53
1. THE CHEMICAL SHIFT:
Soon after the discovery of the phenomenon of nuclear magnetic resonance sci
entists realized that more than a single line was observed for protons in a
organic molecule such as ethanol. In liquid (isotropic) phase, this splitting of
lines is caused by the chemical shift due to the Zeeman interaction and to sca
lar spin,spin couplings.
As we have seen before, the resonance frequency depends on the strength of
the applied static ﬁeld and on the gyromagnetic ratio of the nucleus:
ω = γB
However, the ﬁeld strength to be considered is not exactly the strength of the
applied magnetic ﬁeld in vacuo but is locally modiﬁed by the electronic envi
ronment of the nucleus. What is really important is the effective ﬁeld strength at
the nucleus site. Many different mechanisms are known that may inﬂuence the
exact strength of ﬁeld at the nucleus:
σ = σ
dia
(local) + σ
para
(local) +σ
m
+ σ
rc
+ σ
ef
+ σ
solv
Herein is
• σ
dia
the diamagnetic contribution
• σ
para
the paramagnetic contribution
• σ
m
the neighbor anisotropy effect
• σ
rc
the ringcurrent contribution
• σ
ef
the electric ﬁeld effect
• σ
solv
the solvent effect
1.1 The diamagnetic effect:
The nucleus is surrounded by an electron cloud. The static magnetic ﬁeld B
o
causes the electrons to precess about the axis of the magnetic ﬁeld. Thereby, a
current is created that itself builds up a magnetic ﬁeld. The direction of the
induced ﬁeld is opposed to the static ﬁeld and acts to decrease the strength of
the latter:
LECTURE COURSE: NMR SPECTROSCOPY
Fourth Chapter: Chemical shifts and scalar couplings Pg.54
This effect is called the Lamb Shift. It can be calculated as
in which ρ(r) is the electron density, r the distance of the electron from the
nucleus, e the charge and m
e
the mass of the electron. One important statement
of this socalled BiotSavart Law is that the electron density at larger distances
from the nucleus is of importance for the counteracting ﬁeld.
The diamagnetic shielding describes the behavior of spherically distributed
electrons such as electrons from the sorbital of protons. It is therefore the dom
inant term for proton shifts but less important for the heavier nuclei.
1.2 The paramagnetic term:
The paramagnetic term serves to correct for the disturbance of the spherical
rotation of electrons. This may be caused by the formation of bonds. Quantum
mechanically speaking, the paramagnetic term may be calculated by ﬁnding
the way by which the electron wave functions are modiﬁed by the magnetic
ﬁeld. Such a modiﬁcation of the wave function takes place because properties
FIGURE 1.
B
o
σ
iso
µ
0
e
2
3m
e
⁄ rρ r ( )
0
∞
∫
• dr =
LECTURE COURSE: NMR SPECTROSCOPY
Fourth Chapter: Chemical shifts and scalar couplings Pg.55
of exited states (contributions from the LUMO, lowest unoccupied molecular
orbital) are mixed into ground state wave (esp. the HOMO, highest occupied
molecular orbital) functions through application of the static ﬁeld. The electron
distribution in such an excited state wavefunction is very different from that
encountered in the ground state giving rise to a large change in the shielding:
The calculation of the diamagnetic shift is rather involved and requires the
exact knowledge of the excited wave functions. For
13
C it is given as
In this formula, ∆E is the energy difference between HOMO and LUMO , r the
radius of the 2p orbital and the Q describe electron densities and bond orders.
Very lowlying groundstate wavefunctions are usually paired with rather high
lying excited state wave functions such that the resulting energy separation is
comparably large. Since the paramagnetic shift is inversely proportional to the
energy gap, the shift is large when the gap is small. Reactivities of compounds
are often related to HOMO/ LUMO energy separations and chemical shifts
have therefore been successfully used to screen for reactive compounds.
The paramagnetic term requires nonspherical electron distribution (a non
vanishing angular momentum) and can hence only be applied to nuclei that
have nons orbital electrons. It is the dominant term for all nuclei other than
protons. The shift range due to the paramagnetic contribution is larger than for
the diamagnetic shift. This is obvious from the huge shift ranges (e.g. several
thousand ppm for
57
Fe compared to approx. 15 ppm for
1
H).
Substituents inﬂuence the chemical shift because they change the electron den
FIGURE 2. Mixing of excited state wavefunction into groundstate wavefunctions through the Bo field.
HOMO
LUMO
B
o
σ
i
para
µ
0
µ
B
2
( ) 2π∆E r
3 –
〈 〉 ⁄ Q
i
Q
j
i j ≠
∑
+ =
LECTURE COURSE: NMR SPECTROSCOPY
Fourth Chapter: Chemical shifts and scalar couplings Pg.56
sity at the proton nucleus via inductive effects. This is fairly evident from a
comparison of a oxygen and a carbon bound proton:
1.3 Chemical shift anisotropy:
The distribution of the electrons about the nucleus is usually nonspherically.
The magnitude of the shielding therefore depends on the relative orientation of
the nucleus with respect to the static ﬁeld. For sp
3
carbons the nuclei are tetra
hedrally coordinated and the electron distribution is almost invariant under
rotation. For sp
2
carbons such as aromatic or carbonyl nuclei the shielding
highly depends on the orientation of the bond relative to the static ﬁeld:
The chemical shielding is described by the chemical shift tensor, which trans
forms the static ﬁeld into the effective ﬁeld (see add. mat.) The zz axis is usu
ally taken as the axis with the largest shielding. Due to the shielding the
direction of the effective ﬁeld B
eff
may deviate from the direction of the static
FIGURE 3. Dependence of chemical shielding upon orientation of carbonyl bond toi the external field
FIGURE 4. Shielding of carbonyl carbon in dependence of orientation of zzaxis w.r. to external field
C
H
e = 2.5 e = 2.1
H
O
e = 3.5
e = 2.1
ca 12 ppm
ca 4 ppm
C
O
σ
zz
C
O
σ
zz
LECTURE COURSE: NMR SPECTROSCOPY
Fourth Chapter: Chemical shifts and scalar couplings Pg.57
ﬁeld B
o
. Since the molecule rotates quickly in solution the observed chemical
shift is a average over the shifts coresponding to the different orientations (iso
tropic chemical shift). However, when rotation is hindered, as is the case in the
solid state, the line is signiﬁcantly broadened. In liquids this effect contributes
to relaxation, and is the major relaxation mechanism for nonprotonated car
bons. Chemical shift anisotropy values can be measured from solidstate NMR.
The value of the chemical shift for the isotropic case can be taken as 1/ 3 of the
trace
1.4 Magnetic anisotropy of neighboring bonds and ring current shifts:
Some types of neighboring bonds create an additional magnetic ﬁeld which is
anisotropic in space. An example is a triple bond. The π electrons of the triple
bond form an electron cloud that extends around the bond axis in form of a
tube. The magnetic ﬁeld forces the electrons to rotate about the bond axis creat
ing a magnetic ﬁeld whose direction is along the bond axis and which again
counteracts the static ﬁeld. A similar counteracting ﬁeld is formed in the π
cloud of aromatic systems. Such anisotropies can dramatically change the
appearance of proton spectra. They usually increase the dispersion of proton
spectra. However, in order to give substantial effects the inﬂuenced protons
FIGURE 5. Left: Coordinate system used for the chemical shift tensor. The zz axis points along the value
of the largest shielding, xx and yy orthogonal to zz. Right: Direction of the static and the effective field.
σ
zz
σ
yy
σ
xx
B
o
B
korr
B
eff
σ
iso
1 3 σ
xx
σ
yy
+ σ
zz
+ ( ) ⁄ =
LECTURE COURSE: NMR SPECTROSCOPY
Fourth Chapter: Chemical shifts and scalar couplings Pg.58
must be sterically ﬁxed in relative to the anisotropic group. This is the case for
folded proteins that do adopt a unique structure. Therefore, one can sometimes
judge from the signal dispersion of 1D proton spectra whether a protein is
folded or random coil.
The observed shifts can be either upﬁeld or downﬁeld, depending on the
exact postion of the proton. The following ﬁgure shows regions of increasing
(+) or decreasing () ﬁeld:
These effects can be dramatically. Aliphatic Protons that are ﬁxed in space
above the plane of an aromatic ring can be shifted to values below 0 ppm,
those in the plane to values higher than 10 ppm.
FIGURE 6. Fig:Left: Anisotropy from a benzene π system. Right: Anisotropy from a triple bond. The direction of
the induced electron flow is indicated below
FIGURE 7. Chemical shift anisotropies of various πsystems
e
B
o
C
e
B
o
C
C
C
C
C


+
+


C
C
+++
+


++
+
+




LECTURE COURSE: NMR SPECTROSCOPY
Fourth Chapter: Chemical shifts and scalar couplings Pg.59
The chemical shift anisotropy for a triple bond can be calculated as
where theta is the angle with respect to the bond axis, r the distance to the cen
tre and χ the magnetic susceptibilities parallel and perpendicular to the bond
axis. As can be seen from the formula, the effect depends strongly on the dis
tance and orientation.
1.5 Electric ﬁeld gradients:
Strongly polar groups create intramolecular electric ﬁelds. This has the effect of
distorting electron density in the rest of the molecule and will hence inﬂuence
the chemical shifts.
1.6 Hydrogen bonds:
Hydrogen bonds decrease the electron density at the involved proton site and
hence leads to a highfrequency shift. The effect is especially pronounced for
symmetric hydrogen bonds (those in which the distance of the proton is equal
to both acceptors). Protons that are hydrogen bonded usually be easily recog
nized from their shift. Their shift is highly temperature, concentration and sol
vent dependent. Protons that are part of hydrogen bonds do exchange much
more slowly with labile solvent deuterons and can therefore be differentiated
from others. This is used in protein NMR to identify βsheets or α−helices that
display extended hydrogen bond networks.
FIGURE 8. The hydroxyl group at the top of the molecule is part of a sym. Hbond and will therefore be
downfield shifted.
∆σ 1 3r
3
χ
II
χ
⊥
– ( ) 3 θ cos ( ) [ ]1 4π ( ) ⁄ ⁄ =
O
O
HO
H
LECTURE COURSE: NMR SPECTROSCOPY
Fourth Chapter: Chemical shifts and scalar couplings Pg.60
1.7 Solvent effects:
It is very important to realize that different solvents may have different effects
on the chemical shifts. Quite often an overlapping signal can be resolved when
changing the solvent. Suitable solvents for causing large changes in chemical
shifts are benzene or acetone, which may completely change the appearance of
a spectrum that has been acquired in chloroform. This effect is especially pro
nounced when benzene selectively complexes parts of the molecule due to the
ringcurrent effect from the aromatic ring. Such selective effects may be the
case when not all parts of the molecule can be accessed by the solvent.
1.8 Shifts due to paramagnetic species:
Paramagnetic compounds have unpaired electrons. When paramagnetic impu
rities are contained in the sample, the lines are usually considerably broadened
for solvent excessible protons. However, the chemical shift can also be inﬂu
enced. The mechanism is Fermicontact interaction (see section scalar cou
plings) between the nuclear spin and the unpaired electron. This effect is
moderate, since electron density of the unpaired electron at the observed
nucleus (which is usually not in the same molecule) is required. Since relax
ation times of electrons are very short, the splitting due to the electron,proton
coupling is removed rapidly (selfdecoupling), so that comparably sharp lines
can be observed. Paramagnetic reagents, also known as shift reagents, serve to
disperse proton spectra. Thereby, a 2 ppm shift range for aliphatic protons can
be dispersed over 6 ppm after addition of the shift reagent.
2. SCALAR COUPLINGS:
Two types of interaction between spins are known:
• dipolar coupling
• scalar coupling
The contributions from dipolar coupling usually cannot be observed in isotro
pic (liquid) phase. This is so because the dipolar coupling depends on the ori
entation of the connecting vector to the static ﬁeld. This orientation rapidly
changes in solution due to molecular tumbling and the dipolar coupling there
LECTURE COURSE: NMR SPECTROSCOPY
Fourth Chapter: Chemical shifts and scalar couplings Pg.61
fore averages to zero. However, it can be observed in the solid state or in liquid
crystals.
The scalar coupling leads to a splitting of resonance lines. The effect is medi
ated via the electrons and its magnitude therefore rapidly decreases when the
number of intervening bonds increases.
The basic mechanism that propagates the coupling is the Fermicontact inter
action. This effect describes the coupling between the nuclear and the electron
spin:
Electrons that occupy an orbital that has a nonvanishing electron density at
the nucleus (such as selectrons) have the electron spin antiparallel to the
nuclear spin (Hund's rule). Is the atom bonded to another atom, then the two s
orbitals of the separate atoms form a σorbital, which is occupied by two elec
trons with antiparallel spin (Pauli principle). The second nuclear spin can now
have its spin parallel or antiparallel to the spin of the second electron depend
ing on whether it is in the α or β state. The antiparalell alignment is energeti
cally favourable and hence gives a signal at lower frequency. The magnitude of
the coupling is expressed as the scalar coupling constant and can be calculated
as:
where γ
A
and γ
B
are the gyromagnetic ratios of the involved nulcei,  Ψ
A
(0)
2
is
the electron density at the site of the nucleus A and c
A
the coefﬁcient of the
contribution of the atomic orbital A to the σ MO. ∆
T
is the triplet excitation
FIGURE 9. Spin states of nuclear (red) and electron (blue) spins for hydrogen atom (a) and singlet hydrogen
molecule (b) and triplet hydrogen (c) with corresponding energy diagram.
E
∆E= hν = hJ
a
b
c
a
b
c
J 2µ
0
g
e
µ
B
3 ⁄ ( )
2
γ
A
γ
B
Ψ
A
0 ( )
2
Ψ
B
0 ( )
2
c
A
2
c
B
2
1 ∆
T
⁄ ( ) =
LECTURE COURSE: NMR SPECTROSCOPY
Fourth Chapter: Chemical shifts and scalar couplings Pg.62
energy that measures the energy required to promote an electron from the sin
glet into the triplet spin state (parallel alignment):
The scalar coupling constant is usually given the symbol
n
J, in which n indi
cates the number of intervening bonds. As we will see later, the magnitude of
coupling depends on the amount of overlap of the involved electron orbitals.
The scharacter of orbitals depends heavily on the hybridization of the
involved nuclei. This is very clear from the
1
J
1
H,
13
C coupling constants which
can be roughly categorized as:
•sp
3
: 140 Hz
•sp
2
: 160Hz
•sp: 250 Hz
The sp orbital has the highest content of scharacter and hence gives the largest
coupling.
The coupling also depends on the gyromagnetic ratios of the involved nuclei.
When hydrogen is substituted by deuterium, the corresponding coupling to
another proton is scaled by the factor γ
D
/ γ
H
=1/ 6.514 . Therefore, the couplings
are sometimes reported in form of the reduced couplings, which are deﬁned
as:
2.1 Direct couplings (
1
J):
In order to be able to detect scalar spin,spin couplings it is necessary that the
two protons are not isochronous, meaning that they don’t have the same chem
ical shift. The onebond coupling constant usually has a positive sign.
FIGURE 10. Singlettriplet transition
∆
T
κ
ik
J
ik
h ⁄ ( ) 2π γ
i
⁄ ( ) 2π γ
k
⁄ ( ) =
LECTURE COURSE: NMR SPECTROSCOPY
Fourth Chapter: Chemical shifts and scalar couplings Pg.63
2.2 Geminal couplings (
2
J):
Geminal proton couplings largely depend on the hybridization of the involved
carbon nuclei:
To be able to observe these couplings the geminal bonded protons need to be
diastereotopic in order to give separate proton frequencies.
2.3 Vicinal couplings (
3
J):
Vicinal couplings display a characteristic dependence upon the involved dihe
dral angle:
according to the relation:
3
J= 7  1.0 cosφ + 5 cos2φ
for protons.
For
1
H,
13
C couplings a similar equation has been suggested:
3
J= 3.81  0.9 cosφ + 3.83 cos2φ
This relationship, also known as the Karplus relation, has been successfully
exploited to determine the stereochemistry of compounds. Of course, it
FIGURE 11. Various geminal couplings
FIGURE 12. Left: Karplus curve, right: Definition of dihedral angles
H
2
C
H
H
109
12.4 Hz
H
H
120
H
H
120 H
2
C=C
 4.3 Hz
+2.5 Hz
H
H
b
a
3
J (Hz)
φ
φ/rad
LECTURE COURSE: NMR SPECTROSCOPY
Fourth Chapter: Chemical shifts and scalar couplings Pg.64
requires the dihedral angle to assume a certain value ﬁxed over time. There
fore, rapid averaging of dihedral angles (rotatable bonds) leads to averaging of
coupling constants, which for that case corresponds to the arithmetic mean
from the mainly populated rotational states, e.g. gauche and trans rotamers.
Openchain aliphatic compounds therefore mostly display vicinal couplings of
about 7 Hz.
For systems where the bond cannot be rotated (e.g. cyclic systems or macro
molecules with deﬁned secondary structure such as proteins), the coupling is
very useful to determine the stereochemistry or derive structural constraints:
For example, in sugars the vicinal coupling between the anomeric proton and
the neigbouring proton determines whether the sugar is α or β.
The reason for the dependence of the vicinal coupling on the dihedral angle is
the follwoing: The scalar coupling is propagated via electrons. The angular
dependence comes into play, because the dihedral angle determines how large
the overlap of the molecular orbitals and thereby the efﬁciency of transfer is:
FIGURE 13. Vicinal couplings of systems with hindered rotation about CC bonds
FIGURE 14. Overlap of adjacent orbiatals for φ=0° (left) and φ=90° (right).
3
Ja,e = 25 Hz
3
Ja,a = 1013 Hz
3
Je,e = 25 Hz
3
Ja,b ≈ 11 Hz
3
Ja,b ≈ 1819 Hz
H
2e
H
1a
H
2a
R
H
a
R'
H
b
R
H
a
H
b
R'
φφφ φ
LECTURE COURSE: NMR SPECTROSCOPY
Fourth Chapter: Chemical shifts and scalar couplings Pg.65
Further effects on the vicinal couplings are:
• electron negative substituents decrease
3
J
• increasing HCC bond angles decrease
3
J
• increasing CC bond lengths decrease
3
J
2.4 Longrange couplings:
4
J couplings are rarely observed. For saturated systems they require the atoms
to be coplanar and in zigzag (W) conformation:
2.5 Couplings involving π electrons:
In principle, π electrons cannot propagate scalar couplings because π orbitals
have nodes at the position of the nuclei. Therefore, spincorrelation between σ
and π electrons is required:
Since π electron systems are highly delocalized, these interactions can be prop
agated over many bonds. The orientation of the double bond in
4
J couplings is
not important, e.g. cisoid and transoid couplings are of similar magnitude.
2.6 The number of lines due to scalar spin,spin couplings:
In principle, each coupling doubles the numbers of lines. However, when cou
plings are of similar magnitude, some lines overlap. In the absence of geminal
couplings (no diastereotopic protons) all couplings are usually due to vicinal
FIGURE 15. Systems with
4
J couplings.
FIGURE 16. Scalar couplings involving σ,π transfers
H
2e
H
4e
H
H
H
H
H
H
H
H
C C
H
σσσ σ
πππ π
LECTURE COURSE: NMR SPECTROSCOPY
Fourth Chapter: Chemical shifts and scalar couplings Pg.66
protons. In openchain compounds single bond dihedral angles are rotation
ally averaged so that all vicinal couplings are around 7 Hz (vide supra)´. In this
case the total number of lines of a single resonance due to the coupling with N
neighbouring protons is N+1. In cyclic or structurally welldeﬁned compounds
where the couplings may be much different the total number is 2
(N1)
.
The intensities of the lines in the case of overlapping lines (similar magnitude
of couplings) can be derived from the coefﬁcient in the Pascal triangle:
FIGURE 17. Coupling of a proton a with two other protons b and c for the case of different (left) or
similar (right) couplings.
Number of coupled nuclei Number of lines rel. intensities
I=1/ 2 I=1
0 1 (singlet) 1
1 2 (doublet) 1:1
2 3 (triplet) 1:2:1
3 4 (quartet) 1:3:3:1
4 5 (quintet) 1:4:6:4:1
5 6 (sextet) 1:5:10:10:5:1
6 7 (septet) 1:6:15:20:15:6:1
0 1 (singlet) 1
1 3 (triplet) 1:1:1
2 5 (quintet) 1:2:3:2:1
3 7 (septet) 1:3:6:7:6:3:1
J
ab
J
ac
J
ac
J
ab
J
ac
J
ac
Ω
a
Ω
a
LECTURE COURSE: NMR SPECTROSCOPY
Fourth Chapter: Chemical shifts and scalar couplings Pg.67
2.7 Strong coupling:
The rules for line multiplicities and intensities described above are only valid
in the case of weak coupling. However, when the chemical shift difference is
not very large compared to the scalar coupling constant (∆δ> 10 J), these rules
do not apply anymore. In the strongcoupling case spin properties are mixed.
A practical consequence is that a resonance line cannot be referred to belong to
a spin A or B. Whereas in the limit of weak coupling a spinﬂip of spin A does
not cause spin B to ﬂip there is a probability to do so in the strong coupling
case. In order to derive coupling constants or chemical shifts from strongly
coupled spins these parameters may not be extracted from the spectrum but
have to be derived from a simulation and comparison to the measured spec
trum. For the case of two dublets due to two strongly coupled protons the
inner lines are larger then the outer ones ("roof effect"). Thereby it is possible to
decide which signals are coupled with each other.
FIGURE 18. Second order effects depending on the chemical shift difference. The simulations were calculated using
ν
A
−ν
B
/J=15(1), 3(2), 1(3) and 0(4). In the case of the ABX system the Xpart is shown separately.
4.0
1
2
3
4
4.8 4.9 5.0 ppm 4.8 4.9 5.0 ppm ppm
AB−System
(J =10Hz)
AB
ABX−System
(J =10Hz, J =6Hz, J =4Hz)
AB BX AX
LECTURE COURSE: NMR SPECTROSCOPY
Sixth Chapter:Relaxation Pg.68
1. RELAXATION:
As stated in the ﬁrst chapter thermal equilibrium is a state in which the popu
lation of α and βstates corresponds to the Boltzmann distribution. Further
more, the spins are uncorrelated in phase such that no transverse
magnetization exists. However, when RF pulses are applied the state of the
spins is pertubated away from equilibrium distributions. The process that
brings the spins back to thermal equilibrium is called relaxation:
It is important to note that a particular spin can be only in either the α or the β
state. What relaxation describes is the behaviour of magnetization which is
deﬁned as the sum of the magnetic moments from all spins. However, to
understand the mechanisms of relaxation we will look at the fate of individual
spins.
Phenomenologically, relaxation is categorized into
1. longitudinal relaxation (or T1 relaxation) that describes the return of the z
component of longitudinal (z)magnetization to its equilibrium value. The
corresponding time constant of that process is called T1.
2. transverse relaxation (or T2 relaxation) that describes the decay of trans
verse (x,y) magnetization. Analogously, the corresponding time constant is
called T2.
1.1 T1 relaxation:
The equilibrium distribution of α vs. β state is governed by the Boltzman dis
tribution:
FIGURE 1. left: equilibrium state. right: nonequilibrium state, phases are correlated
B
o
=z
y
x
B
o
=z
y
x
#( )
#( )
( ) / α
β
α β
=
− −
e
E E kT
LECTURE COURSE: NMR SPECTROSCOPY
Sixth Chapter:Relaxation Pg.69
Since α and β states correspond to different energies changing the relative
populations of these states changes the energy of the system. Therefore, T1
relaxation is an enthalpic process. The energy is transferred to or taken from
other spins. The surrounding of spins is often called the lattice and hence T1
relaxation is often referred to as spinlattice relaxation:
Note that the vectors shown in the ﬁgure above represent the magnetization
vectors rather than magnetic moments from individual spins. Longitudinal
magnetization decays exponentially according to
The T1 relaxation time determines the pulse repetition rate, the delay that has
to be inserted between individual scans. T1 times for protons are in the range
of 0.5 to a few seconds. T1 times for quadrupolar nuclei (I > 1/ 2) can be rather
short (ms range). Degassing the sample will remove paramagnetic oxygen
which otherwise facilitates T1 relaxation. Therefore, degassing samples is use
ful when measuring small NOE effects.
1.2 T2 relaxation:
The transverse relaxation determines how fast phase coherence between spins
is lost. Since dephasing in the spins does not cause any changes in the relative
population of α− and βstate, it is not changing the energy of the system. T2
relaxation is merely a entropic process. It is often referred to as spin,spin
relaxation:
FIGURE 2. Return of the zcomponent of magnetization to the equilibrium value. The initial state may
have been created through population inversion arising from an 180˚ pulse.
y
x
y
x
y
x
y
x
y
x
z z
z z z
M t M t e
z z
t T
( ) ( )
/
=
−
0
1
LECTURE COURSE: NMR SPECTROSCOPY
Sixth Chapter:Relaxation Pg.70
Once the transverse relaxation time T2 passed by transverse magnetization has
decayed to 1/ e of its original value:
The transverse relaxation time determines the linewidths of the signals. The
shorter T2 the broader the signals. The linewidth at half height of the signals
can be expressed as:
There is no reason to sample the signal for times much longer than T2 because
only noise will then be added to the FID. The T2 time and hence the linewidth
also determines the theoretically achievable resolution.
1.3 The mechanisms of relaxation:
In principle the spin system may return to equilibrium via stimulated relaxa
tion (some kind of energy exchange process that initiates relaxation) or by
spontaneous relaxation. It can be shown that the probability for spontaneous
relaxation is far too low to account for it.
Mechanistically, relaxation is caused by additional ﬁelds that happen to have
exactly the frequency that corresponds to the required transition. For example,
T1 relaxation takes place if spins change from the α to the βstate or vice versa.
FIGURE 3. Top: Dephasing of transverse coherence leading to T2 relaxation. Lower: Influence of T2
relaxation on the appearance of the FID.
X
y
X
y
X
y
X
y
X
y
X
y
∆t
t
I
Det
t
e
 t/T 2
I
Det
M t M t e
x y x y
t T
, ,
/
( ) ( ) =
−
0
2
∆ν
π
1 2
2
1
/
=
T
LECTURE COURSE: NMR SPECTROSCOPY
Sixth Chapter:Relaxation Pg.71
The energy difference between these states correspond to a speciﬁc frequency
and this frequency matches to a ﬁeld ﬂuctuating at
There are two major mechanisms by which these ﬂuctuating ﬁelds are formed:
• dipoledipole interaction between two different spins
• chemical shift anisotropy
Both processes are connected to the movement of spins in space due to overall
tumbling of the molecules or due to internal motions.
Dipoledipole induced relaxation:
Spin A close in space to spin B causes a (small) dipole moment at the nucleus B:
The induced local ﬁeld can be expressed as:
The magnitude of the induced ﬁeld depends on both the distance of the two
interacting nuclei and on the angle formed between the interatomic vector and
the static ﬁeld. Through molecule tumbling the angle φ permanently changes
its magnitude causing a ﬂuctuating ﬁeld.
For protons that relax mostly through dipole,dipole interaction with other pro
tons the transverse relaxation time T2 and thereby its linewidth is determined
by a) how far the next protons are in space and b) the overall tumbling time of
the molecule. It is often found that isolated protons (those that do not have
other protons on the neighbouring carbons) display incorrect integrals in 1D
proton spectra. This is due to the fact that T1 relaxation for these protons is
FIGURE 4. Left: definition of the vector connecting two spins and its orientation φ with respect to the external field.
Right: Overall tumbling changes φ.
∆E E E h = − =
α β
ν
ν
γ
π
=
B
2
B
o
φ
1
H
15
N
H
N
B
o
φ
r
B
r
loc
=
− µ φ ( cos ) 3 1
2
3
LECTURE COURSE: NMR SPECTROSCOPY
Sixth Chapter:Relaxation Pg.72
inefﬁcient and therefore these protons do not have completely relaxed in
between different scans. The linebroadening of signals due to slow overall
tumbling is obvious from the linewidth of spectra from large proteins that dis
play very broad lines.
The distance between geminal protons depends on the bond geometries but
for all other protons it is determined by the structure. An important application
therefore is the NOE effect, a method that depends on relaxation properties of
spins and helps to elucidate the threedimensional arrangement of protons (see
next chapter).
1.4 Other relaxation mechanisms:
Any particular event that will cause a ﬂuctuating ﬁeld at the site of the nucleus
may contribute to relaxation provided that it delivers spectral density at any of
the possible NMR transitions.
1.4.1 Chemical shift anisotropy (CSA):
Nonspherical distribution of electron density (e.g. occurring for sp
2
hybridized carbon nuclei) will cause a ﬂuctuating ﬁeld upon rotation of the
nucleus which serves as a efﬁcient source for relaxation:
The efﬁciency of CSA relaxation increases with the square of the magnetic
ﬁeld and is the major relaxation source for amide nitrogen nuclei at ﬁelds
higher than 600 MHz. It is also the major relaxation source for nonproton
bearing carbon nuclei such as carbonyl or oleﬁnic carbons.
1.4.2 Scalar relaxation:
Relaxation may also be induced when the scalar spin,spin coupling to another
nucleus ﬂuctuates rapidly. This could be caused by
• chemical exchange of the coupled nucleus (scalar relaxation of the first kind)
FIGURE 5. Dependence of electron shielding for carbonyl carbons on the orientation of the external field relative to
the p
z
orbital
C
O
σ
zz
C
O
σ
zz
LECTURE COURSE: NMR SPECTROSCOPY
Sixth Chapter:Relaxation Pg.73
• rapid T1 relaxation of the coupled nucleus (scalar relaxation of the second
kind).
Scalar relaxation of the ﬁrst kind is observed for protons coupled to quickly
exchanging hydroxyl protons. Scalar relaxation of the second kind is fre
quently encountered for protons coupled to quadrupolar nuclei. An practical
example is the case of nitrile carbons in which the carbon is mostly coupled to
the quadrupolar
14
N nucleus and is usually signiﬁcantly broadened. Scalar
relaxation is much more likely to effect T2 than T1 relaxation times.
1.4.3 Quadrupolar relaxation:
Nuclei with a spin > 1/ 2 rapidly relax under the inﬂuence of a electric ﬁeld
gradient. T1 and T2 relaxation is very fast for these nuclei provided that the
ligand environment of the nucleus is nonsymmetrical. This is seen for
14
N,
which gives a narrow resonance in the tetrahedral NH
4
+
ion but much broader
lines in asymmetrical environment such as pyrrole.
1.4.4 Spinrotation relaxation:
Rapidly rotating groups such as methyl groups induce a ﬂuctuating ﬁeld at the
frequency of rotation. This effect is usually of minor importance.
1.4.5 Interaction with unpaired electrons:
The interactions can be either of dipolar or scalar nature. The effects are dra
matic such that small contaminations with paramagnetic impurities may
severely broaden the lines. Therefore, oxygen (which is paramagnetic,diradi
cal) is often removed through thaw and freeze cycles to increase T1.
For
13
C
NMR relaxation reagents like Cr(acac)
3
are added in very small quanti
ties to bring the T1's down to about 1s (otherwise, carbonyl nuclei may have
T1's of 2030sec) to allow faster pulsing.
1.5 The motional properties:
In this section we will see how the dependence on the motional properties is
expressed mathematically. The timedependence of the motions is of prime
importance since it determines the frequency at which the induced local ﬁelds
ﬂuctuate and therefore whether they may contribute to relaxation or not. In
order to describe how the angle φ changes with time we use the correlation
function g(τ):
that describes how quickly a function changes with time. The following ﬁgure
displays the correlation function for functions with no (left), little (middle) or
g f t f t ( ) ( ) ( ) τ τ = +
LECTURE COURSE: NMR SPECTROSCOPY
Sixth Chapter:Relaxation Pg.74
rapid (right) time dependence:
Since the NMR signal decays exponentially (this can be derived from the line
shape which must be lorentzian for exponential decay), g(τ) is believed to be
exponential:
τ
c
is the correlation time which is deﬁned to be the time required for a 360˚
rotation of the molecule. A very approximate formula valid for globular pro
teins in water is τ
c
≈10
12
M (M = molecular weight in Dalton) but depends
highly on temperature, shape etc.
We are not interested so much into the time it takes for a molecule to change its
spatial arrangement but rather on the frequency with which these changes take
place. Therefore, the spectral densities, which are the Fourier transform of the
correlation function, are preferred. The spectral density J(ω) tells us how much
power is available from the motion of the molecule to cause ﬂuctuations at the
frequency ω. It can be calculated as:
The dependence of the spectral densities on the frequencies are shown in the
ﬁgure below for large (left), mediumsized (middle) and small molecules
(right). It is clear that the densities at low frequencies increase with increasing
correlation times (larger molecules). This can be rationalized in the following
way: The overall power from all frequencies (the integral under the spectral
density function) must remain the same for all molecules. However, smaller
molecules may rotate faster than larger molecules so that the highest possible
frequency is lower for larger molecules.
FIGURE 6. typical correlation functions for no decay (left), slow decay (middle) and fast decay (right.
g e
c
τ
τ τ
( ) =
− /
J
c
c
( ) ω
τ
ω τ
=
+
2
1
2 2
LECTURE COURSE: NMR SPECTROSCOPY
Sixth Chapter:Relaxation Pg.75
The motional fate of a molecule can be described by the following scenario: A
molecule rotates with a certain frequency. Then it hits another molecule and
the rotation is slowed down or stopped. After the crash the molecule starts to
rotate again until it has reached its highest possible frequency or has hit
another molecule:
Small molecules therefore posses a broad band of rotational frequencies
whereas the larger molecules have more spectral density at the lower frequen
cies (in fact, the highest spectral density is at frequency zero). We will see later
that the frequencies that match possible NMR transitions determine how
quickly a nucleus may relax.
1.5.1 The dependence of the relaxation rates on the fluctuating fields in x,y or z
direction:
An additional ﬁeld in zdirection means that the spins additionally precess
about the zaxis with the frequency of that ﬁeld.In contrast, ﬁelds in x or y
directions cause spins to ﬂip from the αto the βstate or vice versa.
FIGURE 7. Spectral density functions for no decay (left), medium decay (middle) and fast decay (right).
FIGURE 8.
H
N
H
N
ν
H
N
H
N H
N
ν ν ν
LECTURE COURSE: NMR SPECTROSCOPY
Sixth Chapter:Relaxation Pg.76
The longitudinal relaxation time depends on the frequency of α to βstate
transitions and therefore on the ﬁelds along x or y:
In order to be effective these ﬁelds must precess with frequencies comparable
to the larmor frequency about z. This is analogous to the case of the B
1
ﬁeld
which can only inﬂuence spins when the ﬁeld precesses about the zaxis.
The transverse relaxation time is determined by the amount of dephasing of
transverse magnetization. Obviously, ﬁelds along z change the precession fre
quency since they alter the strength of the static ﬁeld (which is also along z):
This mechanism is represented by the ﬁeld term along z in the equations
above. Dephasing at J(0) is effective because if the zﬁeld would ﬂuctuate, say
from the +zdirection to the zdirection, the dephasing effect from it would be
reversed and averaged out. It can therefore only be effective if that ﬁeld stays
in the +z direction and hence the spectral density at zero frequency is impor
tant. In addition, rotations about the x or yaxis (causing α to βtransitions)
will reduce the lifetime of the state (lifetime broadening). This can be seen from
the Heisenberg principle which states that the shorter the lifetime is the larger
the uncertainty in energy is. The contribution of lifetime broadening to T2 is
the same as to T1 since it uses the same mechanism and hence similar spectral
densities are required.
FIGURE 9.
B
o
=z
y
x
1
1
2 2 2
T
H H J
x y
∝ + γ ω ( ) ( )
1
0
1
0
2
2 2 2
2
2 2 2
T
H J H J
T
H J H J
x
z y
y
z x
,
,
( ( ) ( ))
( ( ) ( ))
= +
= +
γ ω
γ ω
LECTURE COURSE: NMR SPECTROSCOPY
Sixth Chapter:Relaxation Pg.77
1.6 Excurs: The LipariSzabo model for motions:
The LipariSzabo model (also sometimes referred to as the “modelfree” approach) factorizes
the autocorrelation function into a correlation function for overall tumbling and one for inter
nal motions:
The overall correlation is assumed to decay exponentially
The correlation function describing internal motions is described as
The generalized order parameter S
2
describes the spatial restriction of the internal motion
with respect to a moleculeﬁxed coordinate system. τ
e
is the effective correlation time for that
internal motion. A value of 1 means that a particular vetor does not move relative to the
molecular frame and a value of zero indicates that movement of a vector is completely uncor
related to overall tumbling which is the case for highly ﬂexible loop regions in globular pro
teins for example.
The spectral densities in this model is expressed as:
The strength of the LipariSzabo interpretation is that it gives an impression about the rela
tive “ﬂoppiness” of parts of the molecule in absence of any speciﬁc model about the exact
nature of the internal motion. Assuming free diffusion in a cone the halfangle θ expressing
the spatial restriction of the internal motion can be calculated as
which translates for an S
2
of 0.4 into a half angle of 45°:
g t ( ) g
o
t ( ) g
i
• t ( ) =
g
o
t ( ) e
t τ
R
⁄ –
=
g
i
t ( ) S
2
1 S
2
– ( )e
t τ
e
⁄ –
+ =
J ω ( ) S
2
τ
R
1 ωτ
R
+ ( )
2
⁄ ( ) 1 S –
2
( )τ
e
1 ωτ
e
+ ( )
2
⁄ + =
S
2
θ
max
( ) 0.5 θ
max
( ) 1 θ cos +
max
( )
2
cos =
LECTURE COURSE: NMR SPECTROSCOPY
Sixth Chapter:Relaxation Pg.78
1.7 The nature of the transitions:
So far we have only been talking about a single spin changing his state due to
interaction with the surrounding spins. A transition that involves a single spin
going from the α to the βstate is called a singlequantum transition:
Alternatively two spins may simultaneously go from the α to the βstate (dou
blequantum transition). Furthermore one spin may go from the α to the β
state and another one from the β to the αstate in a correlated fashion (zero
quantum transition). The frequency for the double quantum transition corre
sponds to the sum of the frequencies of the individual spins whereas the zero
quantum frequencies are formed from the difference of their corresponding
frequencies. For homonuclear spin systems the zeroquantum frequencies are
very low (kHz range) and the double quantum frequencies very high.
For T1 and T2 processes spectral densities at frequencies corresponding to
different transitions are utilized. It can be seen from the ﬁgure below that for
T2 relaxation spectral density at J(0) is important in contrast to T1 and NOE. As
we have seen before J(0) steadily increases with increasing molecular size and
for larger molecules such as proteins almost all spectral density is at J(0).
Hence T2 times are very short for large molecules leading to very broad lines.
FIGURE 11. left: Ispin single quantum transitions. Right: Sspin single quantum transitions
FIGURE 12. zero and double quantum transitions
single quantum transitions
αα
αβ
ββ
βα
i Spin
αα
αβ
ββ
βα
SSpin
αα
αβ
ββ
βα
zeroquantum transition
αα
αβ
ββ
βα
double quantum transition
LECTURE COURSE: NMR SPECTROSCOPY
Sixth Chapter:Relaxation Pg.79
Note that the parameters in the ﬁgure below are given for
15
N relaxation data
(the mechanism is dipolar relaxation with
1
H). For
1
H relaxation, the zero
quantum frequencies (which are the difference of the chemical shifts) are in the
010 KHz range and therefore very close J(0) whereas for
15
N nuclei the zero
quantum frequencies are not so much away from the proton frequencies (MHz
range).
For
15
N magnetization, relaxation is mainly due to
1
H,
15
N dipolar interactions
and
15
N chemical shift anisotropy. It depends on spectral density of single
quantum proton coherences, single quantum nitrogen coherences and hetero
nuclear (
15
N,
1
H) double and zeroquantum coherences:
in which ∆ denotes the chemical shift anisotropy of nitrogen. Note, that the
rates of T1 and T2 relaxation differ in the contribution of J(0) for T2. Fore large
molecules, J(0) contains almost all spectral density. Hence, T2 relaxation is very
fast, and this is the principal reason why large molecules display very broad
nmr lines.
FIGURE 13. Dependence of T1, T2 and NOE upon spectral densities at various frequencies.
T1
T2
NOE
o
ω
N
ω
N

ω
H
ω
H
ω
N
+
ω
H
1
T
1
= R
N
(N
Z
) =
γ
H
2
γ
N
2
h
2
4 r
N,H
6
J (ω
H
− ω
N
) + 3 J (ω
N
) +
6 J (ω
H
+ ω
N
)

¦


¦

+
∆
2
ω
N
2
3
J (ω
N
)
1
T
2
= R
N
(N
x ,y
) =
γ
H
2
γ
N
2
h
2
8 r
N,H
6
4 J (0 ) + J (ω
H
− ω
N
) + 3 J (ω
N
) +
6 J (ω
H
) + 6 J (ω
H
+ ω
N
)

¦


¦

+
∆
2
ω
N
2
3
2
3
J (0 ) +
1
2
J (ω
N
)

¦


¦

LECTURE COURSE: NMR SPECTROSCOPY
Sixth Chapter:Relaxation Pg.80
The dependence of transverse and longitudinal relaxation upon the correlation
time is shown in the next ﬁgure. T2 continuously gets shorter with increasing
molecular size due to the increase in spectral density at J(0). T1 however passes
a minimum. Following the point where spectral density at the zeroquantum
frequencies decreases T1 is getting larger again. (T1 relaxation does not depend
upon spectral density at J(0):
1.8 Measurement of relaxation times:
T1 relaxation times are usually determined from the inversion recovery
sequence: 180 deg pulse…delay…90 deg pulse…delay ..acquisition
FIGURE 14. Dependence of T1 and T2 upon the overall tumbling time
T1
T2
LECTURE COURSE: NMR SPECTROSCOPY
Sixth Chapter:Relaxation Pg.81
In this sequence, magnetization is inverted by application of a 180 degree
pulse. A delay follows during which T1 relaxation takes place bringing the z
magnetization back towards +z. Afterwards a 90 degree pulse along y turns
the magnetization onto the xaxis into observable signal. The magnitude and
sign of the xmagnetization at the end of the sequence depends on a) the T1
constant and b) the relaxation delay ∆.
Transverse relaxation time is determined from a CarrPurcell Spin echo. In
principle, one could simply measure the decay of transverse magnetization fol
lowing a 90 degree pulse. However, inhomogeneity of the static ﬁeld leads to
accelerated transverse relaxation. Usually, one is not interested into the contri
bution from ﬁeld inhomogeneity (this would mean that the transverse relaxa
tion time would depend on the quality of shimming) and therefore the Carr
Purcell sequence that eliminates the additional T2 relaxation due to ﬁeld inho
mogeneity is applied:
FIGURE 15. Inversion recovery.
y
x
z
y
x
z
z
z
y
x
z
z
180 deg
90 deg
90 deg
∆t
2∆t
LECTURE COURSE: NMR SPECTROSCOPY
Sixth Chapter:Relaxation Pg.82
In this sequence 180 degree pulses are applied during the T2 relaxation delay
equally spaced by delay periods. The effect of the 180˚ pulses is to refocus
differential precession due to ﬁeld inhomogeneity. The sequence is also
referred to as an spinecho.
For both experiments, the inversion recovery and the CarrPurcell sequence a
set of 1D (or 2D) spectra is recorded with different relaxation periods. The
intensities of the remaining signal is measured and plotted against the
relaxation delay. Mostly these plots yield decaying exponentials and by ﬁtting
procedures (least squares ﬁt to the experimental data) it is possible to extract
the time constant for the decay:
FIGURE 16. CarrPurcell spin echo.
y
x
z
z
90 deg
y
x
z
y
x
z
y
x
z
z
∆t
1
8
0
d
e
g
∆t
LECTURE COURSE: NMR SPECTROSCOPY
Sixth Chapter:Relaxation Pg.83
FIGURE 17. Analytical formula to describe the decay of longitudinal (left) and transversal (right) magnetization.
I
n
t
e
n
s
i
t
y
0.4 0.8 1.2 1.6 2.0
[sec]
I
n
t
e
n
s
i
t
y
[msec]
80 160 240
I(t) = A e
R1/t
+ B I(t) = A e
R2/t
+ B
T1
T2
LECTURE COURSE: NMR SPECTROSCOPY
Seventh Chapter: The NOE Effect Pg.84
1. THE NUCLEAR OVERHAUSER EFFECT (NOE):
If one resonance A is irradiated, an increase (positive NOE) or decrease (nega
tive NOE) of signal intensity of other resonances such as resonance C is
observed when spin C is close in space to spin A:
This phenomenon is called nuclear Overhauser effect or NOE. The NOE effect is
the method for elucidation of 3D structural features and stereochemistry using
NMR together with information from scalar spin,spin couplings. The NOE
enhancement factor η is deﬁned as
in which I and I
o
is the observed intensity of the resonance of spin I with and
without irradiation of spin S, respectively. The efﬁciency through which the
NOE is transferred from spin I to spin S depends strongly on the distance of
the two protons involved and on the tumbling properties of the molecule:
Because of the distance dependence, the NOE is the major tool for elucidation
of stereochemistry of molecules in solution. The NOE depends on the relax
ation rates of zero and double quantum transitions:
σ
IS
is the crossrelaxation rate. Physically speaking, it characterizes the rate of
FIGURE 1. Irradiation of resonance A leads to a increase of peak intensity of the neighboring spin C (positive NOE)
or to a decrease of peak intensity (negative NOE).
Reference
positive NOE
negative NOE
A B
C
A
C
B
η = =
−
( )
f S
I I
I
I
o
o
{ }
η τ =
( )
−
f r
c
6
f S
I
S
I
IS
IS
{ } =
γ
γ
σ
ρ
LECTURE COURSE: NMR SPECTROSCOPY
Seventh Chapter: The NOE Effect Pg.85
dipoledipole transitions that give rise to the NOE. It can be expressed as:
Therein, W
2QC
and W
ZQC
are the rates for doublequantum and zeroquantum
transitions.
ρ
IS
is the longitudinalrelaxation rate. It is the rate constant for direct dipolar
relaxation of spin I by spin S. It corresponds to the rate of recovery of Isignal
after selective inversion. In a twospin system is ρ
IS
equals to 1/ T1. In multi
spin systems, however, dipolar relaxation with other spins contributes to 1/ T1
and then ρ
IS
is not equal to 1/ T1. In the case of multispin systems
ρ
IX
corresponds to dipolar interaction with all other spins X and ρ* to other
relaxation mechanisms for spin I such as CSA. The important point to note
here is that the magnitude of a NOE between two particular spins not only
depends on their distance (and on the tumbling of the molecule) but also on
the fact how many other protons are in close distance:
This is nicely demonstrated in the ﬁgure above which shows the magnitude of
the NOE between two protons A and B. The smaller the angle α the closer the
third spin C is and the more efﬁcient dipolar relaxation of spin A by spin C
becomes thereby dramatically reducing the magnitude of the NOE between
spins A and B. Note that the distance r
AB
remained unchanged!
FIGURE 2. Dependence of the strength of the NOE on spin A caused by irradiation of spin B on the bond angle α.
σ
IS QC ZQC
W W = −
2
R
T
IS IX
x
I 1
1
1
= = + +
∑
ρ ρ ρ
*
f
A
{B}
ααα α
LECTURE COURSE: NMR SPECTROSCOPY
Seventh Chapter: The NOE Effect Pg.86
1.1 Experiments to measure NOEs:
In principle, a NOE enhancement can be triggered whenever the population of
α and β states of a spin system are different from the equilibrium value. This
can be accomplished by
• an inversion pulse (180 degree pulse) that inverts the relative
populations of α and β states
• saturation of a resonance line that causes the corresponding α and β
states to be equally populated.
Utilizing these two different means of disturbing the populations of spin sys
tems the experiments for measuring NOEs can be categorized into the follow
ing
• the steadystate NOE experiment, in which a resonance is selectively
irradiated with low power for a time τ
m
sufﬁciently long to completely
saturate the transition and propagate the NOE (approx. > 0.5s). A
reference spectrum is recorded without irradiation and subtracted. The
difference spectrum shows only signals which have received a NOE
(that means, which have changed their intensity) and the irradiated
resonance.
• the transient NOE. In this experiment a resonance is selectively inverted
with a selective 180˚ pulse. After a delay τ
m
for NOE buildup a 90˚ read
pulse is applied and a 1D acquired. Again, one experiment with and one
without the selective 180˚ pulse are subtracted from each other. The
famous NOESY (or ROESY) is the 2D variant.
FIGURE 3. Left: Equilibrium population. Middle: Population after selective Ispin inversion pulse. Right:
Population after saturation of the Ispin resonance.
I
I
S
S
αα
βα
ββ
αβ
I
I
S
S
I
I
S
S
LECTURE COURSE: NMR SPECTROSCOPY
Seventh Chapter: The NOE Effect Pg.87
1.2 The steadystate NOE:
The steady state NOE depends on the geometry of all protons nearby, not only
on those saturated and observed. The enhancement is:
In this equation the ﬁrst term represents the direct contribution to the NOE.
The magnitude due to the direct NOE is inﬂuenced by nearby protons because
they have an inﬂuence on T1.
The second term in the equation above describes the indirect NOE transfer.
The indirect transfer is a 3spin effect. It is also called spindiffusion. In spin
diffusion a relay spin that is close to both spins serves to transfer the NOE.
Depending on the correlation time and the sign of the NOE one distinguishes
two cases:
1.2.1 Extreme narrowing (η
max
>0):
• Most enhancements are positive but some can also be negative, depending on
the geometry.
• T1 and T2 values are very similar.
• The lines are rather sharp (hence the name extremenarrowing).
• The inﬂuence of the indirect effect is smaller but noticeable. This is
shown in the ﬁgure below that displays the contribution due to the 3
spin effect depending on the angle α (extreme narrowing).
FIGURE 4. Pulseschemes for measuring of steady state NOE (left) and transient NOE(right).
τ
m
τ
m
Ref
NOE
steadystate
tr ansi ent noe
f S
r
r r
f S r
r r
I
IS
IS IX
x
X IX
IS IX
x
x
{ }
{ }
max max
=
+
−
+










−
− −
−
− −
∑ ∑
∑
η η
6
6 6
6
6 6
LECTURE COURSE: NMR SPECTROSCOPY
Seventh Chapter: The NOE Effect Pg.88
1.2.2 Spindiffusion (η
max
<0):
• In the negative NOE regime (large molecules), all enhancements are negative.
• The T2 values are very much shorter than T1.
• The lines are broad.
FIGURE 5. Contributions of direct and indirect (spin diffusion) effects to the overall NOE.
FIGURE 6. Steadystate enhancements depending on the correlation time for a fourspin system. The
relative distances between spins A,B,C and D are 1:2:1 as indicated in the insert. Resonance B is
irradiated.
ααα α
f
C
{A}
direct direct
indirect
total
A
B
C
D
1
1 2
NOE
f
A
{B}
f
C
{B}
f
D
{B}
ωτ
C
LECTURE COURSE: NMR SPECTROSCOPY
Seventh Chapter: The NOE Effect Pg.89
Spindiffusion is very effective and steadystate NOE measurements are completely
useless. This is shown in the ﬁgure above. When the molecules have gained a
certain size the spindiffusion effect spreads the NOE out to all other protons
(see the magnitude of the NOE for ωτ
c
> 100) respectable of what their distance to
the irradiated proton is!
In order to circumvent the problems associated with spindiffusion the trun
cateddriven NOE has been used, in which the irradiation time is limited to
short periods. In both regimes, the extreme narrowing and the spindiffusion
limit, enhancements are nonsymmetrical due to possible relaxation leakage
with a third spin:
1.3 The transient NOE:
In the transient NOE experiment a resonance is inverted by application of a
selective 180 degree pulse or unselectively in a 2D experiment (NOESY). The
NOE enhancement is:
The transient NOE has some features that are remarkably different from the
steadystate NOE:
• Enhancements are symmetrical
irrespective whether spin S has another proton close in space (which quenches
the NOE in the steady state case dramatically).
• Using short mixing times NOE information is still useful in the spindiffusion
case. Spindiffusion can be recognized from the buildup curves of the NOEs (a
number of NOESY experiments are recorded with increased mixing times.
Spindiffusion cross peaks should show a characteristic induction phase).
• 1D transient and NOESY experiments give identical enhancements.
The following ﬁgure shows the transient NOE buildup upon irradiation of
f S f I
I S
{ } { } ≠
f S
D
e e
D R R R R R
I
R D R D
I S IS I S
m m
{ }
( ) ' (
( ' ) ( ' )
)
= −
( )
= − + = +
− + − −
σ
σ
τ τ
1
4
1
2
2 2
f S f I
I S
{ } { } =
LECTURE COURSE: NMR SPECTROSCOPY
Seventh Chapter: The NOE Effect Pg.90
Lys56 NH of bovine phospholipase A
2
and observation of Ala55 NH (N), Lys
56 β
1
H and β
2
H depending on the used mixing time for molecules in the
extreme narrowing (τ
c
= 3x10
11
or 3x10
10
) or in the spindiffusion limit (τ
c
=
3x10
9
or 3x10
8
):
In the extreme narrowing the spindiffusion peak β
2
H is of negligible inten
sity. In the spindiffusion limit it can still be distinguished from the genuine
NOE peaks at short mixing times. This is in contrast to the use of steadystate
NOE's in that case.
1.4 The state of the spin system and the density matrix:
The density matrix describes the state of the spinsystem. A onespin system
may be in the α or β state or may undergo a transition from the α into the β
state (or vice versa). The density matrix contains the probabilities for a given
state or transition and hence is a convenient way to simultaneously represent
all possible states of a spin system. The density matrix contains diagonal ele
ments which correspond to populations. These are states that do not undergo
transitions. Offdiagonal elements are transitions. In thermal equilibrium all
offdiagonal elements are zero.
A twospin system may be in the αα, the αβ, the βα or the ββ state or may
undergo transitions between them. The
FIGURE 7. Strength of the spindiffusion peak (β2) in dependence of the correlation time.
τ
C
=3 x 10
11
τ
C
=3 x 10
10
τ
C
=3 x 10
9
τ
C
=3 x 10
8
NOE
LECTURE COURSE: NMR SPECTROSCOPY
Seventh Chapter: The NOE Effect Pg.91
transitions are called singlequantum coherences, since only one spin changes
its state. Those are the only transitions which lead to observable signals during
detection periods.
The double quantum coherences:
and the zeroquantum coherences (ﬂipﬂop transitions):
are not directly observable but play a very important role for the theoretical
description of the nuclear Overhauser effect (NOE) because their efﬁciency
determines the crossrelaxation rate. Double quantum transitions involve tran
sitions where two spins ﬂip the same way and in zeroquantum transitions the
two spins undergo opposite ﬂips. Double quantum coherences evolve with the
sum of the chemical shifts of the comprising spins, zero quantum coherences
evolve with the difference of their chemical shifts:
FIGURE 8. Occupied states of the density matrix (left) of populations and singlequantum transitions with
corresponding transitions.
αα αβ αα βα
αβ αα βα αα
→  → 
→  → 
or or
or
αα ββ ββ αα →  →  or
αβ βα βα αβ →  →  or
αα
αβ βα ββ
αα
αβ
βα
ββ
Populations
αα
αβ
ββ
βα
αα
αβ
ββ
βα
SingleQuantum Transitions
ISpin SSpin
αα αβ βα ββ
αα
αβ
βα
ββ
LECTURE COURSE: NMR SPECTROSCOPY
Seventh Chapter: The NOE Effect Pg.92
1.5 The sign of the NOE:
The sign of the NOE can be positive (small molecules, extreme narrowing) or
negative (large molecules, spindiffusion limit). The dependence on the size of
the molecule (to be more speciﬁc: on their correlation time) comes into play
because the crossrelaxation rate is deﬁned as:
For proton,proton NOEs the zeroquantum frequencies are close to zero and
the doublequantum frequencies are very high. As we have seen before large
molecules have spectral density only at frequencies close to zero. Therefore,
large molecules relax via zeroquantum transitions whereas small molecules
mostly use doublequantum transitions, hence the change in the sign of the
NOE:
FIGURE 9. Occupied states of the density matrix (left) of zero and doublequantum transitions with corresponding
transitions.
αα
αβ
ββ
βα
ZeroQuantum Transitions
αα
αβ
ββ
βα
DoubleQuantum Transitions
αα αβ βα ββ
αα
αβ
βα
ββ
αα αβ βα ββ
αα
αβ
βα
ββ
σ
IS QC ZQC
W W = −
2
LECTURE COURSE: NMR SPECTROSCOPY
Seventh Chapter: The NOE Effect Pg.93
This is the reason, why NOE spectra give very disappointing results for
medium sized molecules (approx. 2000 m.wt. at RT). At this size efﬁciency of
zeroquantum and doublequantum transitions is approximately the same and
therefore the crossrelaxation rate (which is the difference between the two
rates) is close to zero:
The absolute size of zero and doublequantum transitions depends on the
strength of the applied ﬁeld. In the rotating frame NOE (ROE) experiment an
additional ﬁeld, the socalled spinlock ﬁeld, is applied along a transverse axis
(x or yaxis). In the ROE experiment magnetization therefore precesses about
the x (or y) axis with a much lower frequency as the magnetization precesses
about the zaxis in the NOE experiment. Because the B
1
ﬁeld is much weaker
(kHz range instead of MHz range), the zero or doublequantum transitions
have much lower frequencies, and hence relaxation always takes place via the
FIGURE 10. Spectral densities for a small (left) or large (right) molecule. Middle: Max. NOE in
dependence of the correlation time.
FIGURE 11. NOE and ROE dependence on the correlation time.
J(ω)
og ω
J(ω)
og ω
LECTURE COURSE: NMR SPECTROSCOPY
Seventh Chapter: The NOE Effect Pg.94
doublequantum transitions and no zero crossing is observed:
The ROE buildup is about 2.5 times faster than the NOE buildup and hence
shorter mixing times can be used. However the spins relax with T1ρ during the
spinlock time (which is similar to T2) and hence the ROESY cannot be used for
larger molecules due to rapid relaxation during the mixing time. In contrast,
relaxation during the mixing time of a NOESY takes place with T1 which is
long, even for large molecules.
1.6 Why only zero and doublequantum transitions contribute to the NOE
In a twospin system the transitions α
2
>β
2
give rise to the spin2 resonance
and the transitions α
1
>β
1
to the spin1 resonance:
Let us assume that we irradiate the resonance of spin 2:
FIGURE 12. Axis of rotation during NOE (left) and ROE(right) buildup.
FIGURE 13.
x
y
B
o
=z
x
y
B
o
=z
B
1
=y
NOE
ROE
α
1
α
2
α
1
β
2
β
1
α
2
β
1
β
2
α
1
α
2
α
1
β
2
β
1
α
2
β
1
β
2
spin2 tr ansi ti ons
spi n1 tr ansi ti ons
LECTURE COURSE: NMR SPECTROSCOPY
Seventh Chapter: The NOE Effect Pg.95
Due to the irradiation the populations of the two states α
2
and β
2
are equal
ized. Now, this system tries to go back to the equilibrium state. It can do so via
zero, single or double quantum transitions::
However, because irradiation of the spin 2 resonances still keeps the popula
tions of the two α
2
and β
2
states equal leading to β
1
α
2
= β
1
β
2
and
α
1
α
2
= α
1
β
2
.
As can be seen from the following ﬁgure, only zero and doublequantum tran
sitions lead to a change of the intensity of the spin1 transition (and hence to a
NOE effect):
FIGURE 14.
FIGURE 15.
3/3
3/ 6
3/ 6
6/ 6
α
1
α
2
α
1
β
2
β
1
α
2
β
1
β
2
2/ 4
4/ 8
4/8
α
1
α
2
α
1
β
2
β
1
α
2
β
1
β
2
2/ 4
αα αβ αα βα
αβ αα βα αα
α α β β β β α α
α β β α β α α β
→  → 
→  → 
→  → 
→  → 
or or
or SQC
or DQC
or ZQC
( )
( )
( )
1 2 1 2 1 2 1 2
1 2 1 2 1 2 1 2
W
o
W
1
W
1
W
2
zeroquantu m
tr ansiti ons
singlequantum
tr ansi ti ons
doublequantu m
tr ansiti ons
LECTURE COURSE: NMR SPECTROSCOPY
Seventh Chapter: The NOE Effect Pg.96
1.7 Practical tips for NOE measurements:
• the conditions for measurement must be very stable
• (as always true for methods that rely on differences). Especially, the temperature
must be stable. For the same reason, never use spinning for NOE
measurements! Measuring over night or on weekends is preferred because of
less traffic in the building. Optimize the lock power, adjust lock power just
below saturation to give a strong lock signal.
• the mixing time has to be optimized for the molecule size, do not use too long
mixing times in order to avoid spin diffusion.
• Avoid paramagnetic impurities!
• If very small effects should be measured, remove oxygen (degas the sample;
oxygen is a biradical).
• The sample should be concentrated enough but not too concentrated (little lock
signal).
• For observation of NOE's between methyl groups and other protons, irradiate
the methyl group, because relaxation of methyl protons is mainly governed by
the other methyl protons.
• Pay attention to the choice of the solvent. Use a solvent, that gives an intense
lock signal (DMSO, acetone, rather not CDCl
3
or D
2
O if possible), because
than the lock is more stable. D
2
O also has a large temperature shift of the
solvent line, so that the lines easily shift when the temperature is not stable.
• If the NOE is very small, that means if the tumbling time is such that the NOE is
near to the zerocrossing, going from a nonviscous solvent (acetone) to a
viscous solvent (DMSO) or measuring at lower temperatures may increase the
size of the NOE dramatically (note that at low temperatures the danger is high
that the temperature is not stable).
• Use sufficiently long relaxation delays (35 times T
1
).
FIGURE 16. Populations after zeroquantum (left), singlequantum (middle) and doublequantum (right)
transitions and subsequent restoring due to spin2 irradiation.
3/ 3
3/ 6
3/ 6
6/ 6
2.5/
2.5
3.5/
3.5
3.5/
5.5
3.5/
5.5
5.5/
5.5
2.5/
6.5
2.5/
6.5
6.5/
6.5
LECTURE COURSE: NMR SPECTROSCOPY
Seventh Chapter: The NOE Effect Pg.97
Excurs I: The use of NOE data for structure calculation of peptides and proteins
NMR data can be used for structure calculations of proteins up to about 30 kDa presently. The method
uses the fact, that the strength of the NOE between protons depends on their distance. The volume of
the NOESY crosspeaks may be expressed as
The distance between the two protons may vary due to ﬂexibility of the polypeptide chain. The NOE
depends also on the correlation time.
The ﬁrst step in the procedure is to assign the NOESY spectrum completely. Peaks are then integrated.
The next step is to convert NOESY peak volumes into distances, a process known as “calibration”:
These distances will then serve as upper limits to constrain distances between different protons in the
calculated structures. Due to the ﬂexible nature of polypeptides no lower limits are used (except for dis
ulﬁde bridges). The inherently different ﬂexibility of different classes of protons in peptides (e.g. com
pare backbone atoms with methylgroups) is taken into account by applying different calibration
functions for them. Backbone atoms are calibrated with a r
6
dependence, whereas for NOE’s involving
methyl protons a r
4
calibration will be applied. The constant k can be determined on the basis of known
distances (e.g. the distance of sequential amide protons in helices is about 3Å). Additionally, scalar vic
inal coupling constants may be used to constrain torsional angles.
To calculate structures from these upper limits and torsion angle constraints distance geometry algo
rithms have been used in the past. However, nowadays restrained molecular dynamics calculations are
performed. In these calculations, Newtons equation of motion is integrated:
Thereby, the coordinate of a atom at time t can be calculated from the force acting on it. The force itself
is expressed as the negative gradient of the potential energy. The potential energy function is the normal
force ﬁeld expanded by an additional term reﬂecting the nmr constraints:
During a structure calculation a number of (typ. 2050) randomly chosen conformers is energy mini
mized under a simulated annealing protocol. The NMR constrains in the potential energy function will
force the molecule to adopt a structure that is compatible with the NMR data. After the calculation the
2050 separately energyminimized conformers is superimposed for best ﬁt. A resulting narrow struc
ture bundle indicates good convergence of the calculation. This is mostly the case when enough NOE’s
are present to deﬁne the structure. Flexible parts of the molecule usually will display less NOE’s are
less well deﬁned in the resulting structure bundle.
V r
6 –
〈 〉 f τ
c
( ) =
V k d
4 6 –
⁄ =
m
i
t
2
2
d
d
r
i
t ( ) F
i
t ( ) = i 1 … N , , =
U
pot
U
forcefield
U
NMR
+ =
LECTURE COURSE: NMR SPECTROSCOPY
Seventh Chapter: The NOE Effect Pg.98
Excurs II: Use of dipolar couplings to yield structural constraints:
Orientations of structural elements with respect to each other (e.g. the relative orientation of two helices)
is difﬁcult to describe from NMR experiments. Usually, only a few NOE’s between the two elements
may be used. Any misassignment will lead to a large errors in the relative orientation.
Relatively newly, residual dipolar couplings have been used for describe orientations of parts of the mol
ecule that are separated by a large distance. Dipolar interactions between two protons depend on the ori
entation of the vecto connecting the two atoms with respect to the external ﬁeld
Usually, these dipolar interactions are averaged to zero in solution due to rapid tumbling and therefore
rapid changes in the angle f. However, Bax et al. could show, that it is possible to partially orient macro
molecules in solutions containing bicelles (diskshaped vesicles). These pospholipid vesicles orient in
the magnetic ﬁeld such that the membrane normal is perpendicular to the direction of the magnetic ﬁeld.
Marcomolecules in these solution are then partially oriented by simple steric reasons:
The dipolar coupling can be calculated as:
where c
A
and c
B
are the axial and rhombic alignment tensors and S the order parameter. Thereby, a refer
ence frame for all protons (the alignment frame) is available and orientations are possible with respect to
that frame irrespective of the number of intervening bonds. Thereby, parts of molecules that are far apart
may be oriented relative to each other.
Bo
φ
1H
15N
r
B
r
loc
=
− µ φ ( cos ) 3 1
2
3
B
0
φ
χ
A
δ
dip
S B
0
2
( )γ
A
γ
B
r
AB
3 –
χ
A
3
2
φ 1 – ( ) cos ( ) χ
B
2
sin φ 2φ cos ( ) + [ ] • ∝
LECTURE COURSE: NMR SPECTROSCOPY
Fifth Chapter: Chemical and conformational exchange Pg.99
1. CHEMICAL OR CONFORMATIONAL EXCHANGE:
Let us assume that ethanol binds to a target molecule and dissociates of again:
In such a situation the methyl group of ethanol has different chemical environments in
the bound and in the free state. In the bound state it is under the inﬂuence of the ring
current shift of a tyrosine residue for example, and therefore the chemical shift will be
lowﬁeld shifted. In the free state the anisotropy effect due to the ring current shift has
disappeared. Depending on the exchange kinetics the methyl signal will appear as a
single or as two resonances with more or less exchange broadening. Three different
states can be distinguished:
• Slow exchange (two sets of resonances with reasonable sharp lines)
• Fast exchange (a single, sharp line positioned somewhere between the two
lines)
• Intermediate exchange (a single, very broad (or invisible) line.)
The underlying exchange process may also be chemical exchange, such as exchange
between the hydroxyl protons and protons from dissolved water. Alternatively it could
be conformational exchange. All these processes are frequently encountered in NMR.
1.1 Twosite exchange:
Let as assume the following reaction:
The lifetime of state A is:
and of state B is
FIGURE 1. Binding of methanol to protein in the vicinity of a aromatic residue leads to strong shift of methyl signal
C
H3

C
H2

O
H
CH
3
CH
2
OH
+
Me=1 ppm
Me=0.5 ppm
A B
B A
k
k
+
−
 → 
 → 
1
1
τ
A
k =
+
1
1
/
τ
B
k =
−
1
1
/
LECTURE COURSE: NMR SPECTROSCOPY
Fifth Chapter: Chemical and conformational exchange Pg.100
The NMR parameters that characterize both states are a) the chemical shift, b) scalar
spin,spin couplings and c) the linewidth (transverse relaxation time). The NMR time
scale is deﬁned by the relation of the exchange rate with respect to the magnitude of
the accompanying change in the corresponding NMR parameter:
The exchange time scale is deﬁned by the separation of the two signals relative to the
exchange rate. The ratio is inﬂuenced by
• increasing temperature accelerates the exchange reaction and therefore pushes
the system into the fast exchange regime
• increasing static fields increases the line separation and therefore drives the
system into the slow exchange regime.
The effect of chemical or conformational exchange on the chemical shift is shown
below. Therein, a signal in the fast exchange regime is into the slow exchange regime
pushed by decreasing the temperature or increasing the ﬁeld strength. At the coales
cence temperature a very broad line with a plateau is observed.
Exchange rates
Time scale slow intermediate fast
Chemical Shift k << δ
A
 δ
B
k = δ
A
 δ
B
k >> δ
A
 δ
B
Scalar Coupling k << J
A
 J
B
k= J
A
 J
B
k >> J
A
 J
B
Transverse Relax. k << 1/ T
2A
1/ T
2B
k = 1/ T
2A
1/ T
2B
k >> 1/ T
2A
1/ T
2B
FIGURE 2. Appearance of twosite exchange spectra in dependence on the exchange rate. Top: Fast exchange,
Bottom: Slow exchange, 2nd row from top: coalescence.
fast exchange
slow exchange
coalescence
B
o
T
e
m
p
LECTURE COURSE: NMR SPECTROSCOPY
Fifth Chapter: Chemical and conformational exchange Pg.101
1.1.1 Fast exchange:
At fast exchange a single resonance line is observed at the frequency
It is obvious from the equation above that the resonance position of the averaged signal
may not be simply the arithmetic mean of the resonance positions of the two slow
exchanging lines because the population of the two states has to be taken into account
(usually a unknown).
The exchange reaction causes an additional contribution to the linewidth of
Thereby, the faster the reaction or the smaller the frequency difference (provided that a
single line is observed, fast exchange regime) the narrower the line. Such an increase in
reaction rate can be accomplished via a temperature increase and a decrease in fre
quency separation by lowering the magnitude of the static ﬁeld.
A similar situation exist with averaging of couplings:
An important example for averaging of couplings is found for hydroxyl pro
tons of alcohols that exchange with solvent water protons. In the ﬁgure below,
the boxed proton feels the vicinal proton to be in the αstate (left side).
Through chemical exchange that proton is substituted by a proton that comes
from water and is in the βstate. When the exchange is slow both lines of the
doublet are visible but for the fast case only the average singlet will be
observed. This mechanism has been termed selfdecoupling and is the reason
why OH protons in alcohols usually appear as (broad) singlets:
FIGURE 3. Exchange of the hydroxyl proton with a proton from water may lead to a change in the spin state of the
OH proton. If this process is fast, only the avaraged line at the center without splitting due to coupling is visible.
δ δ δ
ave A A A B
p p = + − ( ) 1
∆ν
π ν ν
1 2
2
1
1
2
/
=
− ( )
+
A B
k
J p J p J
ave A A A B
= + − ( ) 1
CD
3
C O
D
H
H O H
H
+
CD
3
C O
D
H
H
H O
H
+
LECTURE COURSE: NMR SPECTROSCOPY
Fifth Chapter: Chemical and conformational exchange Pg.102
1.1.2 The slow exchange limit:
When two separate signals are observed the additional contribution to the linewidth
due to exchange is:
Hence, temperature decrease or ﬁeld increase will lead to a sharpening of the lines.
A typical example where both situations (slow and fast exchange) are simultaneously
encountered is exchange of amide and hydroxyl protons of peptides with solvent in
aqueous solution. At slightly acidic pH the amide protons give separate, sharp lines and
the exchange rate is on the order of 110 sec
1
. In contrast, exchange between hydroxyl
and water protons is so fast that the hydroxyl protons coincide with the water chemical
shift and cannot be observed separately.
1.1.3 The intermediate case:
The lineshape of the signals changes upon passing from the slow into the fast exchange
regime. A detailed investigation shows that it may be described as:
The following ﬁgure shows calculated lineshapes for two signals 10 Hz apart (top
trace) depending on the lifetime (which is the inverse of the reaction rate) or for two
signals with 0.1sec lifetime depending on the frequency separation (given in Hz):
At coalescence the lineshape is characterized by a ﬂat top. The corresponding lifetime
at coalescence is given by
Usually, one is not interested in recording spectra with exchangebroadened lines. You
FIGURE 4. Top: Two lines, 10 Hz separated appear as a singlet, if the lifetime of the separate states is short (0.01s)
or as two separate lines, when the lifetime is long (1s). Bottom: Calculated for a lifetime of 0.1 s with varying
frequency separations (115 Hz).
∆ν
π
=
+
k
1
g
A A X
A X A A X
ν
τ ν ν
ν ν ν π τ ν ν ν ν
( ) =
− ( )
− + ( )






+ −
( )
−
( )
2
1
2
2
2
2 2
2 2
0.2
0.01 0.05 0.085 0.1
0.3 0.5 1.0
5 4
3 1 6 10
15
∆∆∆ ∆δδδ δ=10 Hz
τττ τ
a
∆∆∆ ∆δδδ δ
τττ τ
a
=0.1 sec
τ
π
ν ν
A
c
A X
= −
2
( )
LECTURE COURSE: NMR SPECTROSCOPY
Fifth Chapter: Chemical and conformational exchange Pg.103
can ﬁnd out the proper measuring conditions by
• recording spectra at higher temperature
==>when the lines get sharper heat until the lines are sufficiently narrow. When
that temperature cannot be reached (sample not heatstable or solvent is
volatile), record spectra at lower field.
• when the lines get broader upon temperature rise cool down or proceed at higher
field.
If you cannot get spectra with reasonable sharp lines following this procedure it is
worth trying to change the solvent which may have a dramatic effect.
The following ﬁgure shows 1D spectra (expansions) of chinin recorded at 300 and 600
MHz. It is obvious that by going from 600 to 300 MHz a considerable improvement in
linewidths has been achieved. Often cyclic compounds display such dynamic behavior
due to slow conformational interconversions.
1.2 Investigation of exchange processes:
1.2.1 EXSY spectroscopy:
The 2D EXSY experiment is in principle identical to the NOESY pulse experiment.
However, the mixing time is usually chosen shorter, since the exchange rate is normally
much faster than the crossrelaxation rate. Crosspeaks are due to exchanging (chemical
FIGURE 5. Appearance of spectra displaying exchange phenomena in dependence on the static field
2.0 2.5 3.0 3.5 ppm
600MHz,300K
600MHz,320K
300MHz,295K
300MHz,310K
LECTURE COURSE: NMR SPECTROSCOPY
Fifth Chapter: Chemical and conformational exchange Pg.104
or conformational) protons.
1.2.2 Saturation transfer:
When a signal is inverted (e.g. by application of a selective 180 deg. pulse), it recovers
with T1. However, other protons that are in exchange with the inverted or saturated res
onance become also saturated or inverted:
In this ﬁgure resonance B has been inverted. The signal is shown after successive time
increments for resonance A (left) or resonance B (right). The top trace shows the case
without exchange between A and B (no effect on A). The middle trace displays a dra
matic effect on resonance A when exchange occurs. However, the longitudinal relax
FIGURE 6. 2D EXSY:left: Spectrum overview, right:Expansion
FIGURE 7. Singleline spectra of resonance A (left) and B (right)after various time increments. Top: No exchange.
Middle:Exchange, infinitely long T1, bottom: exchange, T1=1s.
ppm
2 3 4 5 6 7 ppm
2
4
6
ppm
7.0 7.5 ppm
7.0
7.5
A
B
A
B
k
ex
=0
k
ex
=0,T1=inf.
k
ex
=0,T1=1s
LECTURE COURSE: NMR SPECTROSCOPY
Fifth Chapter: Chemical and conformational exchange Pg.105
ation time T1 has been assumed to be inﬁnity. In the lower trace the signal A recovers
due to T1 relaxation.
A frequent case where saturation transfer occurs is through presaturation of solvent.
Hydroxyl protons that exchange with the solvent (e.g. water) vanish from the spectrum
or largely decrease in intensity when solvent presaturation is applied. From the rate of
recovery the exchange rate can be calculated (provided T1 has been determined
before).
1.3 Determination of activation parameters:
The observation of dynamic processes in NMR spectra allows to calculate parameters
for the activation parameters of the exchange process. The activation energy for such a
process can be calculated according to the Arhenius equation
in which K
#
is the equilibrium constant for the formation of the activated complex.
This is related to the reaction rate k via
Theory of thermodynamics states that
so that
therein,
κ is the transmission coefficient (usually 1)
k the Boltzmann constant
T the temperature
h the Planck constant
A plot of ln(k/T) versus 1/T yields a line with slope ( ∆ H
#
/R) and intercept (23.76 + 
∆ S
#
/R).
In close agreement the activation energy can be estimated as
E RT K
a
= − ln
#
k
kT
h
e
G RT
=
−
κ
∆
#
/
∆ ∆ ∆ G H T S
# # #
= −
k
kT
h
e e
H RT S R
=
−
κ
∆ ∆
# #
/ /
∆G RT
T
Jmol
c
c
A B
#
. ln [ ] ≈ +
−
(
\

\
!

−
22 96
1
ν ν
LECTURE COURSE: NMR SPECTROSCOPY
Eighth Chapter: The product operator formalism Pg.106
1. THE PRODUCT OPERATOR FORMALISM (POF) FOR DESCRIPTION OF
PULSEEXPERIMENTS:
Simple onedimensional NMR experiments may be conveniently described
using the pictorial vector description. However, in order to more deeply
understand twodimensional NMR or multiplequantum spectroscopy the
product operator formalism is required.
The following events may happen during or after application of radiofre
quency (RF) pulses:
• chemical shift precession
• evolution of scalar or dipolar couplings
• change of the state of the spins due to additional RF fields
• relaxation
In the following we will see how these events are described in the product
operator formalism (POF). Truly speaking, the POF may only be used to
describe spin systems in the weak coupling case (no second order effects). In the
POF, precession and scalar coupling can be calculated in arbitrary order. If not
necessary, relaxation effects are usually ignored but can be added easily.
In the POF calculations are done on magnetization, which is the ensemble
property formed by adding up all dipole moments from separate spin, rather
than on individual spins. The transformations due to shift precession or cou
pling are described as rotations in threedimensional cartesian space:
We have seen before that a continous rotation can be described as an harmonic
oscillation and presents a superpostion of a sine and cosine modulated func
tion.
FIGURE 1. Rotation of a vector after a time 1/4ν
x
y
x
y
∆t=1/4ν
LECTURE COURSE: NMR SPECTROSCOPY
Eighth Chapter: The product operator formalism Pg.107
In the example given above a property aligned along the xaxis rotates about
the zaxis onto the yaxis with a frequency ω. In the POF this will read as fol
lows:
The symbol over the arrow indicates the axis and the frequency of rotation.
1.1 RF pulses:
Application of a RF ﬁeld along the xaxis will rotate zmagnetization in the z,y
plane:
It is described as:
Of particular importance are rotations of 90 and 180 degrees. The frequency of
rotation depends on the strength of the ﬁeld. The ﬁeld strength is expressed in
terms of the frequency, which is the inverse of the time required for a 360˚ rota
tion.
The sign convention for rotations is that applying a ﬁeld along the xaxis will
rotate in clockwise sense (Field along y: Look down the yaxis and rotate clock
wise, e.g. from z to x)
1.2 Chemical shift precession:
After application of a 90˚ RF pulse spins are correlated in phase leading to a
bunching of phases. The resulting magnetization vector rotates in the x,y plane
FIGURE 2. Rotation of zmagnetization about the xaxis. This will achieve the following conversions: z > y > z
> y > z
A A t A t
x
A
x y
z
ω
ω ω  →  ( ) + ( ) cos sin
B
o
=z
y
x
B
o
=z
y
x
B
o
=z
y
x
I I I
z
I
z y
x
β
β β  →  − cos sin
γ
π
B
PW
1
2
1
360
=
° ( )
LECTURE COURSE: NMR SPECTROSCOPY
Eighth Chapter: The product operator formalism Pg.108
(transverse plane) with a certain angular velocity. After a certain amount of
time it will have rotated by a speciﬁc angle depending on its offset frequency.
Note that chemical shift evolution can only take place for transverse magnetiza
tion!
Shift precession amounts to a rotation of the magnetization vector in the trans
verse plane.
The precession frequency is
ω=2πδ
where δ is the offset from the transmitter frequency.
1.3 Scalar spin,spin coupling:
Due to the fermicontact interaction spins that have a neigbouring spin in the
αstate are of slightly different energy than those that have them in the βstate.
Having different energies means that these two different kinds of spins precess
with different frequencies because frequency and energy are related to each
other. In a reference coordinate system that rotates with the center frequency of
a dublet the downﬁeld component will rotate with a frequency of +J/ 2 in one
direction and the other component with –J/ 2 in the other direction. When both
vectors are 180˚ out of phase the resulting term is
This state is of particular importance for coherence transfer which is the crucial
step for polarization transfer experiments (e.g. INEPT, DEPT) or the mixing
FIGURE 3. Left: Equilibrium state. Middle: State after 90 deg RF pulse. Right: Shift precession in the
transverse plane.
B
o
=z
y
x
B
o
=z
y
x
x
y
I I t I t
x
I
x y
z
ω
ω ω  →  ( ) + ( ) cos sin
I S I S I S S I S
y y y y z
α β α β
+ − = − = ( ) ( ) 2 2
LECTURE COURSE: NMR SPECTROSCOPY
Eighth Chapter: The product operator formalism Pg.109
steps in 2D experiments. In the product operator formalism the transformation
under scalar spin,spin coupling is described as:
A number of operators are frequently encountered during the calculations:
• I
x
, I
y
transverse x (or y) magnetization
• I
z
longitudinal magnetization
• 2I
x
S
z
antiphase I magnetization (or I magnetization antiphase w.r. to S)
• 2I
z
S
z
longitudinal twospin order
• 2I
x
S
y
twospin coherence (mixture of zeroand doublequantum
• coherence, multiplequantum coherence)
Two operators that are both transverse cannot evolve scalar couplings amongst each
other!
FIGURE 4. Vectorial representation of the states due to evolution of scalar couplings
I I Jt I S Jt I
x
J I S
x y z
J I S
x
z z z z
π π
π π  →  +  →  − cos( ) sin( ) 2
x
y
x
y
x
y
I((( (S
ααα α
))) )
x
y
I(S
βββ β
))) )
2IyS
z
x
y
I
x
I
x
Σ=
The product operators can be directly related to the appearance of signals. Scalar
couplings do occur as inphase splitting when the operator is
I
x
cos(πJ
I,S
t)
or as antiphase splitting if the operator is
2I
y
S
z
sin( πJ
I,S
t)
The operator 2IySzsin(πJ
I,S
t)cos(πJ
I,X
t)
leads to the following signal:
J
I,S
J
I,S
J
I,S
J
I,X
LECTURE COURSE: NMR SPECTROSCOPY
Eighth Chapter: The product operator formalism Pg.110
1.4 A simple onedimensional NMR experiment:
A conventional onedimensional experiment for recording proton spectra
looks like the following:
(In the following, 90 degree pulses are drawn as narrow, shaded rectangles, 180
deg. pulses as black, wider rectangles). A 90 degree RF pulse applied along the
xaxis is immediately followed by data acquisition. Using the POF we can sum
marize the events for a 2spin system. Usually, one starts with pure zmagneti
zation. The events are subsequently calculated:
• 90 degree xpulse:
• chemical shift evolution
• scalar spin,spin coupling:
Note that chemical shift and scalar coupling may be calculated in arbitrary
order (but not the effect of pulses and shift/ coupling!).
During a pulse sequence all possible interactions have to be calculated which
may lead to a huge number of terms. However, only a limited number of terms
lead to detectable magnetization at the end and only those are of interest
because they contribute to the signal:
• directly observable are only single operators that contain transverse terms
(I
x
,I
y
, S
x
, S
y
..)
• antiphase magnetization (such as I
y
S
z
) evolves into inphase magnetization
(I
x
) if the corresponding coupling is resolved
• terms that contain more than one transverse operator are not observable and
hence can be ignored in the end of the sequence
FIGURE 5. A simple 1D experiment consisting of a 90° pulse followed by sampling of the FID.
x
I I
z
I
y
x
90( )
 →  −
−  →  − + I I t I t
y
tI
y x
z
ω
ω ω cos( ) sin( )
− +  →  − +
+ +
I t I t I t Jt I S t Jt
I t Jt I S t Jt
y x
Jt I S
y x z
x y z
z z
cos( ) sin( ) cos( )cos( ) cos( )sin( )
sin( )cos( ) sin( )sin( )
( )
ω ω ω π ω π
ω π ω π
π
2
2
LECTURE COURSE: NMR SPECTROSCOPY
Eighth Chapter: The product operator formalism Pg.111
Certain terms that are of negligible intensity can be removed during the calcu
lation thereby largely reducing the number of operators.
1.5 The effect of 180 degree pulses:
180 degree pulses are often used to
• refocus chemical shift
• refocus scalar couplings
When a 180 degree pulse is placed in the centre of a delay it will refocus chemi
cal shift evolution of transverse magnetization (see left part of the following
ﬁgure).
180 degree pulses will ﬂip spins from the α into the βstate:
The effect of 180 degree pulses are summarized in the following ﬁgure:
We have seen before that the two dublet lines arising from spin,spin coupling
are due to different spins that have neighbouring spins in either the α or the β
state. When this coupling partner is ﬂipped from the α into the βstate or vice
versa the coupling evolution will be refocussed after a time that is the same as
the one during which the system was allowed to evolve the coupling. In this
context it is very important to distinguish two cases:
• both spins I and S experience the 180 degree pulse which is usually the case if
they are both protons (homonuclear case)
• only one of the two nuclei experiences the pulse which is the case for a
1
H,
13
C
pair.
In the ﬁrst case both spins are simultaneously ﬂipped and no overall effect
occurs. Only the latter leads to decoupling.
FIGURE 6. Effects of 180° pulse(s) on shift precession and evolution of scalar couplings
I I I I
α β β α 180 180
 →   → 
∆∆∆ ∆
∆∆∆ ∆
I
S
∆∆∆ ∆ ∆∆∆ ∆ ∆∆ ∆ ∆ ∆∆∆ ∆
refocussing of I shift
refocussing of I,S coupling
I shift evolution
refocussing of I,S coupling
refocussing of I shift
evolution of I,S coupling
LECTURE COURSE: NMR SPECTROSCOPY
Eighth Chapter: The product operator formalism Pg.112
The refocussing of chemical shift can be described with the product operator
formalism easily in the following way:
and for evolution of heteronuclear scalar couplings:
However, homonuclear scalar couplings are not refocussed since both spins
are ﬂipped:
1.6 Coherence transfer:
Coherence transfer is the crucial step of
• polarization transfer experiments (INEPT,DEPT)
• multidimensional correlation experiments
In order to transfer coherence couplings must have been evolved. Antiphase I
spin coherence is then transferred into antiphase Sspin coherence:
When the time t during which scalar coupling takes place is exactly equal to
(2J)
1
sin(πJt)=sin(π/2) and Ispin coherence has quantitatively been trans
formed into antiphase coherence. Note that in the homonuclear case steps [2]
and [3] can be achieved with a single pulse. It is also important to realize that
step [2] requires the phase of the 90˚ pulse to be x. Otherwise, multiplequan
tum coherences are formed:
Through the coherence transfer step transverse Ispin magnetization has been
transformed into transverse Sspin magnetization.
I I I I
x y
x
y x
∆ ∆
 →   →  −  → 
180( )
I I S I S I
x y z
S
y z x
∆ ∆
 →   →  −  →  2 2
180( )
( )
I I S I S I
x y z
I S
y z x
∆ ∆
 →   →  − −  →  − 2 2
180( , )
( )( )
I I Jt I S Jt
I S I S a
I S I S a
x
Jt I S
x y z
y z
I
z z
z z
S
z y
z z
x
x
( )
( )
( )
cos( ) sin( )
π
π π  →  + [ ]
 →  [ ]
 →  − [ ]
°
°
2 1
2 2 2
2 2 3
90
90
2 2 2
2 2 3
90
90
I S I S b
I S I S b
y z
I
y z
y z
S
y y
y
x
°
°
 →  [ ]
 →  − [ ]
( )
( )
LECTURE COURSE: NMR SPECTROSCOPY
Eighth Chapter: The product operator formalism Pg.113
1.7 Polarization transfer:
Polarization transfer is a common building block in pulseexperiments to
increase sensitivity. The sensitivity is proportional to the number of spins that
can undergo a certain transition. We have seen before that this number
depends on the relative populations of α and βstates according to the Boltz
mann distribution. The higher the energy gap between the two states the larger
the population difference and hence the stronger the intensity of the signal.
13
C spectra are much lower in sensitivity because
• the natural abundance of
13
C is 1/100 of that of protons
• the energy separation between α and βstates is γ(
13
C)/ γ(
1
H)=1/ 4
and hence the number of transitions are much lower.
Polarization transfer is aimed at bringing the population difference from pro
tons to carbons and hence increasing the sensitivity.
The prototype for the PT sequences is the INEPT experiment:
Using the POF the INEPT sequence can be summarized in the following way:
In principle, one might be tempted to remove the 180˚ pulses on the proton and
carbon channel. However, chemical shift evolution would then take place for
protons transforming
The second term would be transformed into nonobservable multiplequan
tum coherence through the cascade of the following two 90˚ pulses:
Therefore, we need a 180˚ pulse in the center of the proton delay. However, this
FIGURE 7. Pulse sequence for INEPT experiment (
1
H,
13
C polarization transfer).
1
H
13
C
x y
1/4 J(C,H) 1/4 J(C,H)
I I I S I S I S
z
I
y
J I S
x z
I
z z
S
z y
x
y
x
90
1
2
90
90
2 2 2
°
°
°
 →  −  →   →  −  → 
( ) ( , )
( )
( )
2 2 2 I S I S t I S t
x z
tI
x z y z
z
ω
ω ω  →  + cos( ) sin( )
2 2
90
I S t I S t
y z
I S
y y
y x
sin( ) sin( )
( , )
ω ω
°
 →  −
LECTURE COURSE: NMR SPECTROSCOPY
Eighth Chapter: The product operator formalism Pg.114
proton pulse would unfortunately decouple protons from carbons, and we do
need the coupling evolution for the transfer. Hence, a 180˚ pulse on the carbon
channel is additionally required in order to avoid decoupling.
The important achievement of the PT sequence is that the amount of antiphase
carbon magnetization is proportional to the starting Ispin magnetization
which is I
z
=(I
α
I
β
). At the end of the PT sequence the amount of transverse
cabon magnetization depends on the Boltzmann distribution of the proton lev
els which is much more favourable!
An experiment that has become even more popular than INEPT is the DEPT
sequence. The DEPT experiment allows to record
13
C spectra that are edited
with respect to the number of protons that are directly bonded to the carbons.
Nonproton bearing carbons are missing in DEPT spectra. The editing is
achieved through suitable choice of the length of the ﬁnal proton pulse in the
sequence. For β=135 degrees, methyl and methin carbons are positive and
methylen carbons are negative. For β =90 degrees, only CH carbons are con
tained. The dependence of the signal intensity for CH
3
,CH
2
and CH carbons
on the proton pulse length is shown below:
Since CH and CH
3
carbons can usually be distinguished on the base of their
chemical shifts, a DEPT135 experiment is mostly sufﬁcient but should be com
plemented by a DEPT90 in case of ambiguities for CH
3
/ CH. The DEPT
sequence relies on uniform
1
J (CH) coupling constants, a large variation of cou
pling constants leads to crosstalk in the edited spectra.
Of course, a double PT 2D experiment can be used to increase the senitivity
FIGURE 8. Transfer amplitudes for CH
n
(n=13) groups in DEPT experiments in dependency of the flip angle of
the final proton pulse.
45° 90° 135°
CH
2
CH
CH
3
LECTURE COURSE: NMR SPECTROSCOPY
Eighth Chapter: The product operator formalism Pg.115
further such as the HSQC experiment:
FIGURE 9. Comparison of sensitivity of carbondetection experiments without PT ( inversegated
13
C,
13
C{
1
H}), and with PT (INEPT) and with
1
H detection (2*PT, HSQC)
S
e
n
s
i
t
i
v
i
t
y
(
f
u
l
l
y
r
e
l
a
x
e
d
,
1
0
0
%
i
s
o
t
o
p
i
c
a
b
u
n
d
a
n
c
e
)
γ
(
1
3
C
)
5
/
2
γ
(
1
3
C
)
5
/
2
+
N
O
E
γ
(
1
H
)
(
1
3
C
)
3
/
2
γ
(
1
H
)
5
/
2
D
e
c
o
u
p
l
i
n
g
R
D
D
e
c
o
u
p
l
i
n
g
R
D
R
D
D
e
c
o
u
p
l
i
n
g
R
D
t
1
D
e
c
o
u
p
l
i
n
g
1
H
1
3
C
1
H
1
3
C
1
H
1
3
C
1
H
1
3
C
i
n
v
e
r
s
e

g
a
t
e
d
1
3
C
1
3
C
{
1
H
}
I
N
E
P
T
H
S
Q
C
LECTURE COURSE: NMR SPECTROSCOPY
Eighth Chapter: The product operator formalism Pg.116
Excurs: The foundations of the product operator formalism:
The wavefunction for the state of a onespin system can be formulated as
in which the coefﬁcients c
1
and c
2
indicate to which extent the spin occupies the α or
β state. ψ> is called a state vector and α> and β> are the basis states. The basis
states are orthogonal vectors in Hilbert space (a Ndimensional vector space for a
system with N basis states with some special properties). The scalar product of the
two vectors is deﬁned as:
which is the square of the length (norm) of the state vector ψ:
The length of the state vector ψ> is interpreted as the probability of ﬁnding the spin
system in a particular state. The scalar product of two different states is
in which the component printed in bold indicates the amount of interference between
the two states. <ψ'ψ’’> is interpreted as the probability to go from state ψ’ to ψ’’.
We can now deﬁne an operator A that transforms ψ'' into ψ’:
Often in NMR these transformations are rotations in 3D space that do not change the
length of the vectors (socalled “unitary transformations”) and the operator is a rota
tion matrix. Assume we have two state vectors, one describing the inital state of the
spin system, one describing the ﬁnal state:
ψ  〉 c
1
α  〉 c
2
β  〉 + =
ψ ψ 〈  〉
2
c
1
2
c
2
2
+ =
α>
β>
ψ>
C
1
C
2
α>
β>
ψ>
ψ>
‘
“ ψ
tot
>
φ
ψ' ψ'' 〈  〉 ψ' ψ' 〈  〉 ψ'' ψ'' 〈  〉 2 ψ' ψ' 〈  〉
1 2 ⁄⁄⁄ ⁄
ψψψ ψ'' ψψψ ψ'' 〈  〉
1 2 ⁄⁄⁄ ⁄
φφφ φ cos + + =
ψψψ ψ''  〉 A ψψψ ψ'  〉 =
ψ
i
 〉 c
1i
α  〉 c
2i
β  〉
ψ
f
 〉 c
1f
α  〉 c
2f
β  〉 + =
+ =
LECTURE COURSE: NMR SPECTROSCOPY
Eighth Chapter: The product operator formalism Pg.117
To mathematically express the transformation we can arrange the state vector of the
initial state as a column vector and the state vector ofthe ﬁnal state in form of a row
vector and write:
The timeevolution of the density matrix is described by the Liouvillevan Neumann
Equation:
in which H is the Hamiltonian describing the interaction (coupling, shift etc.). The
commutator
The time evolution of the spin states can be expressed as:
The basic rotations are:
c
1i
c
2i
A
11
A
12
A
21
A
22
c
1f
c
2f
=
t d
d
σ t ( ) i H σ t
0
( ) , [ ] – =
i H σ t
0
( ) , [ ] – i Hσ σH – ( ) – =
C C
if C C
or
C C i C C
if C C
q
C
q
q p
q
C
q q p
q p
p
p
 → 
[ ]
=
 →  +
[ ]
[ ]
≠
,
cos , sin
,
0
0
θ θ
Ix
2IySz
2IzSz
z
x
y
z
LECTURE COURSE: NMR SPECTROSCOPY
Ninth Chapter: 2 dimensional NMR experiments Pg.118
1. TWODIMENSIONAL NMR SPECTROSCOPY:
The introduction of twodimensional NMR spectroscopy has largely increased
the potential power of NMR as a tool for structure elucidation of larger mole
cules. It's main advantages are:
• dispersion of signals into two orthogonal dimensions and
• identification of correlations
The 2dimensional NMR experiment is characterized by the introduction of a
second frequency axis, which allows to correlate frequencies:
The two frequency domains are called the direct (F2) and the indirect (F1) fre
quency domains. Frequencies of signals in the direct dimension have been
directly detected in the receiver coil, those of the indirect dimension were
derived from the second Fourier transform of the amplitude modulated sig
nals.
Homonuclear 2D spectra are usually symmetric about the diagonal. The diago
nal contains the onedimensional spectrum. Offdiagonal peaks at the fre
quency F2=Ω
A
, F1=Ω
B
are called crosspeaks, and they indicate the spins with
frequencies Ω
A
and Ω
B
are correlated.
Twodimensional experiments can be classiﬁed into certain groups:
FIGURE 1. Appearance of homonuclear COSY spectrum with crosspeaks (C) and diagonalpeaks(D).
F1
F2
Ω
ΑΑΑ Α
Ω
ΑΑΑ Α
Ω
ΒΒΒ Β
Ω
ΒΒΒ Β
C
C D
D
LECTURE COURSE: NMR SPECTROSCOPY
Ninth Chapter: 2 dimensional NMR experiments Pg.119
• homonuclear correlated spectra that contain frequencies of similar nuclei in
both dimensions (e.g.
1
H,
1
H COSY, INADEQUATE)
• heteronuclear correlated spectra that contain frequencies of different nuclei
(e.g.
1
H,
13
C correlations) in different frequency dimensions.
Similarly experiments can be subdivided into
• shiftcorrelated 2D experiments, that link two spins via their chemical shifts
(F2: chemical shift of spin A, F1: chemical shift of spin B for crosspeaks)
• Jresolved experiments, that separate chemical shift and Jcoupling information
in two orthogonal frequency dimensions (F2: chemical shift of spin A, F1: J
coupling of spin A).
Each pulsesequence for a 2D experiment contains the basic elements
For the example of the F1=Ω
A
, F2=Ω
B
crosspeak the spin A is excited in the
preparation period, then chemical shift labelled during the evolution period.
Subsequently, magnetization is transferred from spin A to spin B in the mixing
period. Finally, magnetization is detected on the spin B.
The 2D data matrix is recorded by performing a set of 1D spectra, in which a
delay called the evolution time is systematically incremented. The signal of the
spin B, which is detected, varies in amplitude from experiment to experiment,
FIGURE 2. Building blocks of 2D experiment.
(Excitation of spin A)
(Chemical Shift labelling of spin A)
(Coherence transfer to spin B)
Detection
Preparation
Evolution
Mixing
LECTURE COURSE: NMR SPECTROSCOPY
Ninth Chapter: 2 dimensional NMR experiments Pg.120
and the modulation frequency of the signal intensity corresponds to the chem
ical shift of the spin A. The 2D spectrum is yielded from a twodimensional
Fourier transformation:
1.0.1 The preparation period:
In principle, the preparation period serves to create transverse coherences. In a
proton,proton correlation experiment like the COSY, it is a simple 90 degree
pulse for excitation. Some experiments that correlate carbon (or nitrogen) with
proton frequencies use an initial INEPT polarization transfer from proton to
carbon to increase sensitivity (dramatically). These experiments are the so
FIGURE 3. Translation of the amplitude modulation of a signal into the frequency of the indirect
dimension through Fourier transform.
LECTURE COURSE: NMR SPECTROSCOPY
Ninth Chapter: 2 dimensional NMR experiments Pg.121
called inversedetection experiments. In the product operator formalism the
preparation is described as
or in the case of inversedetection experiments:
1.0.2 The evolution period:
The evolution period is the pulse sequence element that enables frequency
labelling in the indirect dimension. Usually, the corresponding time is called t1
in contrast to t2 in the direct detection dimension. Fourier transform of the t1
domain data yields the frequency dimension F1 and t2 corresponds to F2. In
order to understand how frequency labelling in the indirect dimension is
achieved, it is useful to recall how the frequencies in the direct dimension are
sampled. The signal is sampled in discontinuous mode:
Consecutive data points are separated by a dwell time. The length of the dwell
time is related to the spectral width (SW, the width of the spectrum in Hz):
The resolution depends on the number of data points sampled and the spectral
width (neglecting relaxation effects).
Frequency labelling in the indirect dimension is done analogously. However,
FIGURE 4. Sampling of data in the direct dimension.
I I
z
x
y
90°
 →  −
I I I S I S
z
I
y
I S
x z
I S
z x
x y y 90 180
90
2 2
° − −
°
 →  −  →   → 
( ) ( , )
( , )
∆ ∆
AQ
DW
(td =16)
dw
SW
=
1
LECTURE COURSE: NMR SPECTROSCOPY
Ninth Chapter: 2 dimensional NMR experiments Pg.122
data points are taken in separate 1D experiments. During a 2D experiment a
series of 1D spectra is recorded which differ by the fact that the evolution time
has been systematically incremented from experiment to experiment. This is
schematically shown in the ﬁgure on the following page for signals that lead to
diagonal peaks (no magnetization transfer during the sequence). The signal
precesses during t1. Depending on the length of the evolution period more or
less of the magnetization will be turned onto the zaxis thereby modulating the
intensity of the remaining signal. Since the evolution time is incremented sys
tematically in successive FIDs the frequency in the indirect dimension is sam
pled analogously to the one of the direct dimension.
In the product operator formalism frequency labelling is described as:
1.0.3 The mixing period:
The many 2D experiments differ in the way they transfer magnetization from
spin A to spin B, that means in the construction of the mixing process. Possible
transfersequences may rely on
• scalar couplings (COSY, TOCSYtype experiments)
• dipolar couplings (NOESY/ROESY type experiments)
• chemical exchanges (EXSY type experiments)
In product operator language the mixing for the COSYtype experiments is
described as
Herein, transverse Ispin coherence is transferred into transverse Sspin coher
ence. Operators like I
x
S
z
evolve with the chemical shift of the spin I whereas
operators like I
z
S
x
evolve with the shift of spin S. Because the mixing step sepa
rates the t1 and t2 periods, such a transfer step will lead to signals correspond
ing to crosspeaks after 2D FT. However, depending on the chemical shift
evolution of the I spin in t1, the term just before the transfer step can also be
I
y
S
z
(instead of I
x
S
z
). Application of a 90° pulse with phase y on the I and S spin
will give I
y
S
x
, a mixture of double and zeroquantum coherence, which cannot
I I t I t
x
t
x I y I
1
1 1
 →  ( ) + ( ) cos sin ω ω
2 2
90
I S I S
x z
I S
z x
y y
°
 → 
( , )
LECTURE COURSE: NMR SPECTROSCOPY
Ninth Chapter: 2 dimensional NMR experiments Pg.123
be observed.
FIGURE 5. COSYtype mixing in the vector model
P
R
E
P
A
R
A
T
I
O
N
M
I
X
I
N
G
D
E
T
E
C
T
I
O
N
E
V
O
L
U
T
I
O
N
F
T
(
F
2
)
1
3
4
5
F
I
D
i
n
F
2
F
I
D
i
n
F
1
2
LECTURE COURSE: NMR SPECTROSCOPY
Ninth Chapter: 2 dimensional NMR experiments Pg.124
The TOCSY transfer is different in the way that inphase magnetization is trans
ferred at twice the rate:
For the NOESY mixing the description is
Some of the more advanced tripleresonance experiments mixing includes
magnetization relay via many nuclei of possibly different nature.
1.0.4 The detection period:
The detection period simply comprises acquisition of the FID with or without
heteronuclear decoupling.
1.0.5 Hetcor and inversedetection experiments:
The heteronuclear correlation experiments (Hetcor, HMQC, etc.) are in princi
ple of the COSYtype. However, the mixing pulse must be applied for both
kinds of nuclei (
1
H and
13
C) separately. Inverse detection experiments include
an additional INEPTtype protonheteronucleus polarization transfer step.
Thereby, the sensitivity is increased according to
In addition, faster pulsing is possible since the proton T1's are usually much
FIGURE 6. Magnetization flow for coherence transfer between spins A and B.
PREPARATION
α
β
MIXING
α
β
α
β
EVOLUTION
Spin A
Spin B
I S
x x
" " 180°
 → 
I S
z z
m
τ
 →  − ( )
Int
ex
∝ γ γ
det
/ 3 2
LECTURE COURSE: NMR SPECTROSCOPY
Ninth Chapter: 2 dimensional NMR experiments Pg.125
shorter than those of
13
C or
15
N.
The prototype inverse detection experiment, the HSQC (heteronuclear single
quantum coherence) experiment is shown below. The experiment contains two
polarization transfer (INEPT) steps.
1.1 Phasecycling:
A large problem in [
13
C,
1
H] spectra is suppression of protons bound to
12
C.
FIGURE 7. Building blocks of homonuclear (left) and heteronuclear (middle and right) experiments.
FIGURE 8. Building block of HSQC experiment.
homonuclear
Excitation
Evolution
Mixing
Detection
heteronuclear
(
1
H detection mode)
PTTransfer back to proton
Preparation
Evolution
Detection
PTTransfer to Xnucleus
Preparation
Evolution
Detection
PTTransfer to Xnucleus
heteronuclear
(X detection mode)
∆ ∆
t
1
∆
∆
DEC
Preparation
INEPT Evolution
ReINEPT
Detection
H
z
H
y
2H
x
C
z
2H
z
C
y
2H
z
C
y
cos(ω
C
t
1
)
2H
y
C
z
cos(ω
C
t
1
) 2H
y
C
z
cos(ω
C
t
1
)cos(ω
H
t
2
)
1
H
13
C
LECTURE COURSE: NMR SPECTROSCOPY
Ninth Chapter: 2 dimensional NMR experiments Pg.126
This is achieved through phasecycling of the ﬁrst 90˚ pulse on the carbon
channel. The satellite lines (those proton lines, which belong to the
1
H
13
C
fragment) are inverted when the phase of the ﬁrst 90˚ carbon pulse is shifted by
180˚. When the receiver phase is inverted too addition of the two FIDs leads to
cancellation of the central proton line (the one that belongs to the
1
H
12
C frag
ment). Clearly, unstable measuring conditions will cause incomplete cancella
tion, and the centre signal will show up as t
1
noise:
1.2 An Alternative: Pulsed Field Gradients
By applying pulsed ﬁeld gradients the homogeneity of the external magnetic
ﬁeld is destroyed and spins with the same chemical shift are defocused to a
degree which depends on their location in the probe. Usually the NMR spec
troscopist spends a considerable amount of time to have maximum ﬁeld
homogeneity over the sample volume. By detuning the zshim starting from a
perfectly shimmed magnet a linear B
0
gradient is created along the sample axis
so that spins in the upper part of the tube have higher frequencies than those at
the bottom. If such a gradient would be applied during signal acquisition a
broad signal corresponding to a wide range of frequencies would be observed
instead of a sharp line. The crucial point is that the amount of dephasing is pro
portional to the coherence level of the spins at the time the gradient is applied.
Doublequantum coherences are dephased by twice the amount than single
FIGURE 9. Coherence selection in HSQC spectra and origin of t
1
noise in HSQC spectra and
1
J
C,H
φ
1
=x
φ
Rec
=x
FID1
φ
1
=x
φ
Rec
=x
FID2
Σ
∆
∆
1
H
13
C
∆ ∆
t1
HSQC
DEC
φ
LECTURE COURSE: NMR SPECTROSCOPY
Ninth Chapter: 2 dimensional NMR experiments Pg.127
quantum coherences, zeroquantum coherences are not dephased at all. Usu
ally at least two gradients are applied during the sequence. In the double
quantum ﬁltered COSY experiment a gradient is applied during the time dou
blequantum coherence is exists. At the end of the sequence (when single
quantum coherences are existent) a readout gradient is used of half the strength
to refocus the gradient. The 2:1 ratio of the two gradients ensures that the
desired coherence pathway has been selected:
The change of the lamor frequency is proportional to the order of coherence p
i
,
the gyromagnetic ratio γ
i
of the spin, the strength and sign of the applied linear
ﬁeld gradient pulse G
z
, it's coordinate Z
i
in the probe and the duration τ:
By applying a gradient of the same strength but opposite sign, coherences of
the same order are rephased again. Alternatively, proton coherences can be
labelled using a pulsed ﬁeld gradient, and, after magnetization transfer to car
bon13, carbon single quantum coherences can be refocused by a gradient of
opposite sign and appropriate strength. Since dephasing and rephasing is done
within the same scan, no further scans are needed provided the S/ N is sufﬁ
cient. Because spectra are recorded without addition/ subtraction of scans.
FIGURE 10. Effect of ﬁeld gradients on isochronous spins at different locations in the probe. (A)
DQFCOSY sequence with application of gradients (B); (C) selected coherence transfer
pathway; (D) vector representation of isochronous spins during the experiment.
t1
BO
Gz
Gz
BO
A
B
C
D
∆υ γ τ ∝ p Z G
i i i z
LECTURE COURSE: NMR SPECTROSCOPY
Ninth Chapter: 2 dimensional NMR experiments Pg.128
instrumental instabilities are less pronounced and therefore twodimensional
experiments tend to contain much less t
1
noise. Furthermore, solvent suppres
sion is superior, no saturation transfer due to fast exchanging protons is possi
ble and signals with identical chemical shifts to those of the solvent can easily
be observed. Whenever dephasing and rephasing intervals are separated by a
long delay, as is the case for NOESY or TOCSY experiments, diffusion leads to
a signiﬁcant loss of magnetization. A second disadvantage stems from the rela
tively long duration of gradients and the time required for reestablishing the
homogenous ﬁeld.
1.3 Hybrid 2D techniques:
Hybrid experiments are very similar to threedimensional NMR experiments,
in which the second time variable is kept constant. Classical examples are
HMQC/ HSQC experiments coupled with a subsequent TOCSY or NOESY
(ROESY) transfer step. In these experiment, not only the direct (
1
J(C,H) connec
tivities are observed, but also connectivies to other (mostly vicinal) protons of
the proton spin system. This is especially helpful in case of overlap of proton
frequencies. One obviously useful ﬁeld for application lies in oligosaccharide
NMR. For example, a [
13
C,
1
H] HSQCTOCSY experiment correlates not only
the anomeric proton with its carbon but also with the neighboring protons.
Obviously, the sensitivity of the correlations is reduced when compared with
the HSQC experiment. For small molecules with small NOE enhancements, a
HSQCNOESY on natural abundance is very insensitive. In case of isotropic
enrichment, such a HSQCNOESY can be a rather sensitive experiment for
larger molecules (those that give strong NOE enhancements).When the mixing
time is limited (e.g. 12ms) coherence transfer in HSQCTOCSY experiments is
limited to neighboring protons. Then, information from such an experiment is
similar to that of COSY spectra but signals are dispersed along the carbon
dimension making it a very valuable experiments for (oligo)saccharides or
peptides.
LECTURE COURSE: NMR SPECTROSCOPY
Ninth Chapter: 2 dimensional NMR experiments Pg.129
1.4 Overview of 2D experiments:
homonuclear shift correlated spectra
via scalar couplings
(DQF)COSY vicinal/ geminal protons correlated
COSYlr COSY optimized for small couplings (longrange
couplings)
INADEQUATEmostly used for carbon, carbon correlation experiments
for nat. abundance samples.
relayCOSY COSY with additional relay step
TOCSY experiment with multiple proton relay, for long
mixing times all protons within the same spinsystem are
correlated
doublequantum spectroscopy
identiﬁcation of degenerate methylene protons
TQFCOSY identiﬁcation of glycine residues in peptides
Xﬁltered Exp. normal 2D experiment, but only spins that have an
additional coupling to a heteronucleus are displayed
via dipolar couplings
NOESY correlation via dipolar interaction
ROESY rotating frame analogue of the NOESY
exchange spectroscopy
EXSY correlates exchanging protons (chemical and conforma
tional exchange)
heteronuclear shift correlated spectra via scalar coupling
FIGURE 11. Building blocks of HSQCTOCSY experiment and information content of the spectra.
∆
∆
1
H
13
C
∆ ∆
t1
H
C
HSQCTOCSY
DEC
spinlock
spinlock
t1
TOCSY
∆ ∆
1
H
13
C
∆ ∆
t1
H
C
HSQC
DEC
Ω
Α
F
1
=
1
3
C
Ω
Α
F2=
1
H
HSQC
C
A
C
B
C
C
H H
H
F2=
1
H
F
1
=
1
3
C
Ω
Α
Ω
Β Ω
C
Ω
Α
HSQCTOCSY
C
A
C
B
C
C
H H H
LECTURE COURSE: NMR SPECTROSCOPY
Ninth Chapter: 2 dimensional NMR experiments Pg.130
13
C detected experiments
hetcor correlation of directly bonded protons and carbons
COLOC proton, carbon correlation via long range (
2
J,
3
J) couplings
1
H detected experiments
HMQC,HSQCcorrelation of directly bonded protons and carbons
HMBC proton, carbon correlation via long range (
2
J,
3
J) couplings
the following experiments include an additional TOCSY/ NOESY/ COSY
transfer step:
H(S)MQCTOCSY
H(S)MQCNOESY
H(S)MQCCOSY
via dipolar couplings
HOESY correlation via heteronuclear dipolar interaction (via
NOE)
Experiments for measurement of Jcouplings
homonuclear jresolved experiments
heteronuclear jresolved experiments
ECOSY
HMQCJ
1.5 Original references for 2D experiments:
COSY:
Aue, W. P.; Batholdi, E.; Ernst, R. R. J. Chem. Phys. 1976, 64, 22292246.
COSYDQF:
Piantini, U.; Sørensen, O. W.; Ernst, R. R. J. Am. Chem. Soc. 1982, 104, 68006801.
Rance, M.; Sørensen, O. W.; Bodenhausen, G.; Wagner, G.; Ernst, R. R.;
Wüthrich, K. Biochem. Biophys. Res. Commun. 1983, 117, 479485.
COSYLR:
Bax, A.; Freeman, R. J. Magn. Reson. 1981, 44, 542561.
E.COSY:
Griesinger, C.; Sørensen, O. W.; Ernst, R. R. J. Chem. Phys. 1986, 85, 6837.
LECTURE COURSE: NMR SPECTROSCOPY
Ninth Chapter: 2 dimensional NMR experiments Pg.131
TOCSY:
Bax, A.; Davis, D. G. J. Magn. Reson. 1985, 65, 355360.
Braunschweiler, L.; Ernst, R. R. J. Magn. Reson. 1983, 53, 521528.
NOESY:
Jeener, J.; Meier, B. H.; Bachmann, P.; Ernst, R. R. J. Chem. Phys. 1979, 71, 4546.
Wüthrich, K. NMR of Proteins and Nucleic Acids; 1st ed.; Wiley: New York, 1986.
INADEQUATE:
Bax, A.; Freeman, R.; Kempsell, S. P. J. Am. Chem. Soc. 1980, 102, 48494851.
Bax, A.; Freeman, R.; Frenkiel, T. A. J. Am. Chem. Soc. 1981, 103, 21022104.
ROESY:
BothnerBy, A. A.; Stephens, R. L.; Lee, J.M. J. Am. Chem. Soc. 1984, 106, 811
813.
Bax, A.; Davis, D. G. J. Magn. Reson. 1985, 63, 207213.
HMQC:
Bax, A.; Griffey, R. H.; Hawkins, B. L. J. Magn. Reson. 1983, 55, 301315.
Müller, L. J. Am. Chem. Soc. 1979, 101, 44814484.
HSQC:
Bodenhausen, G.; Ruben, D. J. Chem. Phys. Lett. 1980, 69, 185189.
HMBC:
Bax, A.; Summers, M. J. Am. Chem. Soc. 1986, 108, 20932094.
LECTURE COURSE: NMR SPECTROSCOPY
Eleventh Chapter: SolidState NMR spectroscopy Pg.132
1. SOLID STATE NMR SPECTROSCOPY:
SolidState NMR spectroscopy is increasingly being used for a number of reasons:
• it allows to probe properties in the solid state (> material sciences)
• it enables to structural characterize insoluble compounds such as (synthetic)
polymers
• it has per se no moleccular size limit and therefore is of advantage when looking
at very large systems (e.g. membraneproteins embedded in bilayers)
However, a number of additional interactions do appear that complicate spectra to an
extend that they become essentially useless unless they are experimentally removed.
Although similar interactions are also encountered in the liquid state they are averaged
out due to rapid tumbling of the molecules. We will therefore shortly review basic
interactions that are important for solidstate NMR applications. The typical range of
various interactions is listed in the table below (from Ref. Williamson et al.):
1.1 The chemical shift
It has been recognized very early on that the resonance frequency of a nucleus is inﬂu
enced by the surrounding electrons. For nuclei with nonspherical electron distribu
tions the shielding effect depends on the orientation of the electron cloud with respect
to the static ﬁeld B
0
. For nonsp
3
hybridized carbons the anisotropy can be as large as
150 ppm and even larger values are encountered for heavier elements.
interaction nuclei typical magnitude
chemicalshift range
1
H ~15 ppm
13
C ~200 ppm
15
N ~200 ppm
anisotropy of CSA
1
H < 10 ppm
13
C < 140 ppm
15
N < 200 ppm
onebond dipolar coupling
1
H
13
C ~22 kHz
1
H
15
N ~20 kHz
13
C
13
C 4.5 kHz
13
C
15
N 2 kHz
15
N
15
N < 1 kHz
LECTURE COURSE: NMR SPECTROSCOPY
Eleventh Chapter: SolidState NMR spectroscopy Pg.133
The chemical shift tensor σσσ σ denotes the tensor required to transform the B
o
ﬁeld vector
into the vector B
eff
representing the effective ﬁeld:
By suitable choice of the coordinate system the chemical shift tensor only contains the
diagonal elements σ
xx
, σ
yy
and σ
zz
. Such a system is called the principal axis system
(PAS). For the shielding tensor the zaxis points into the direction of the largest elec
tron density. The trace of the tensor (by convention σ
zz
≥ σ
xx
≥ σ
yy
)
denotes the isotropic value of the shielding tensor, is rotationally invariant and corre
sponds to the shift observed in isotropic solution.
The parameter δ is the anisotropy and reﬂects the deviation from cubic symmetry
and η the asymmetry, which reﬂects the deviation from axial symmetry:
The orientation of the shift tensor is depicted below:
Beff
x
Beff
y
Beff
z
σ
xx
σ
xy
σ
xz
σ
yx
σ
yy
σ
yz
σ
zx
σ
zy
σ
zz
0
0
B
0
⊗ =
σ σ
xx
σ
yy
σ
zz
+ + ( ) 3 ⁄ =
δ σ
zz
σ
3

–
\ !
( \
=
η σ
xx
σ
yy
+ ( ) δ ⁄ =
z
y
x
O
C
13
13
C
O
O
C
13
B
o
B
o
B
o
z
x
y
x
y
δ
δ
zz
δ
yy
δ
xx
z
LECTURE COURSE: NMR SPECTROSCOPY
Eleventh Chapter: SolidState NMR spectroscopy Pg.134
In a noncrystalline powder sample the nuclei are randomly oriented and the observed
chemical shift tensor corresponds to the statistical average over all possible orienta
tions. The powder lineshapes are characteristic for the magnitudes of anisotropy δ and
asymmetry η. The examples depicted at the left correspond to axially symmetric ligand
ﬁelds. For nuclei with cubic symmetry (e.g.
15
N in NH
4
) even sharper signals are
observed:
Note that in crystalline material orientations are nonrandom! Sample preparation is
very important for solidstate NMR applications:
The ﬁgure above displays
13
C and
15
N MAS spectra of antamanide from a) lyophilized
powder, b) and c) microcrystalline powder obtained by evaporation of solvent with dif
ferent protocols (see ref. Williamson et al.). Structural disorder or inhomogeneity rep
resents a major issue in solidstate NMR applications!
σ
yy
σ
zz
σ
xx
σ
xx
σ
yy σ
yy
σ
yy
σ
yy
σ
yy
σ
zz
σ
zz
σ
zz
σ
zz σ
zz
σ
xx
σ
xx
δ < 0
δ > 0
η=0
η=0
η=0.5
η=0.5 η=1
η=1
10 20 30 40 50 60 70
(a)
(b)
(c)
13
C
15
N
ppm 100 110 120 130 140
LECTURE COURSE: NMR SPECTROSCOPY
Eleventh Chapter: SolidState NMR spectroscopy Pg.135
1.2 Dipolar couplings:
Dipolar couplings depend on the
orientation of the vector that connects the
two nuclei relative to the static ﬁeld, on the
separating distance and on the
gyromagnetic ratios of the two nuclei
involved.
For dipolar couplings the Hamiltonian is:
is homonuclear couplings
H
IS
= d (3 cos
2
β1) I
Z
S
Z
; d = (µ
o
/4π) hγ
I
γ
S
/r
IS
3
and for hetereonuclear couplings:
H
II
= d/2 (3 cos
2
β1) 3I
1Z
I
2Z
 I
1
I
2
; d = (µ
o
/4π) hγ
I
2
/r
II
3
In solution, dipolar couplings are averaged to zero due to rapid (rotational) motion of
the molecules (but give rise to the important relaxation phenomena). In the solid state
no such rapid rotation exists and hence the dipolar couplings give rise to line splittings
which can easily exceed 10 kHz in magnitude. Because these dipolar couplings can be
so large they are not only observed between direct neighboring nuclei but also longer
reaching interactions are encountered. Note that dipolar couplings have recently also
been introduced into solution NMR by the use of weakly aligned systems for recording
residual dipolar couplings (RDCs).
The scalar (J) couplings do not fundamentally differ from the quantities observed in
isotropic (solution) phase. However, it is very important to remove heteronuclear scalar
couplings to increase resolution (and hence sensitivity) in the spectra. RF irradiation
schemes similar to those used to remove heteronuclear couplings in solution are
applied. For spins with I≥1, quadrupolar couplings exist, but those will not be
described here.
Solidstate NMR experiments fundamentally suffer from the additional dipolar interac
tions and from the chemical shift anisotropy. Due to the increased linewidths sensitiv
ity is an important issue in SSNMR, and the quantities required are usually much
larger than for solutionstate NMR.
β
µ
A
µ
B
r
LECTURE COURSE: NMR SPECTROSCOPY
Eleventh Chapter: SolidState NMR spectroscopy Pg.136
1.3 Magic Angle Spinning (MAS)
The effects from chemical shift anisotropy and dipolar couplings lead to excessively
broad lines. To a large extend this linebroadening can be removed by spinning the
sample about the magic angle 54.74° (magicangle spinning, MAS). For this angle the
secondorder legendre polynomial (3cos
2
β1)/2, which describes the angular depen
dence in both the dipolar coupling as well as in the chemical shift Hamiltonians, van
ishes. In MAS, the sample is placed in a cylindrical rotor which is rapidly spun (up to
3050 kHz) about an axis that is tilted by 54° away from the direction of the static ﬁeld.
The tremendous linenarrowing effects due to MAS are displayed in the powder
13
C
spectra of
13
C,
15
N labeled glycine below (from ref. Williamson et al.):
At the lower frequencies spinning sidebands fall into the spectral regions of interest.
Upon rising the spinning frequencies those bands occur outside the spectral region. The
broad lines observed in the powder spectrum collapse into rather sharp lines which
occur at the position of the isotropic chemical shift.
Unfortunately spinning the samples at high frequencies does heat up the sample (tens
of degrees!) and the heating effect must be removed by cooling the rotor appropriately.
1.4 Sensitivity Enhancement:
Solutionstate NMR normally utilizes proton detection experiments due to the high
gyromagnetic ratio of protons which makes this nucleus very sensitive. In SSNMR
applications proton detection is less favorable due to the large number of homonuclear
dipolar interactions, and mostly carbon detection is used instead. Carbon, however, is a
much less sensitive nucleus. Polarizationtransfer techniques are therefore employed to
270 240 210 180 150 120 90 60 30 0 30
δ [ppm]
θ
m
54.7° =
ω
r
B
0
static
5 kHz
10 kHz
LECTURE COURSE: NMR SPECTROSCOPY
Eleventh Chapter: SolidState NMR spectroscopy Pg.137
transfer the much more favorable proton polarization onto the carbon nuclei.
1
Nowadays HartmannHahn Cross Polarization (CP) is used. In this technique B
1
ﬁelds
are simultaneously applied to the proton and carbon spins such that the precession fre
quencies of the two spins are approximately equal (γ
Η
B
H
≈ γ
C
B
C
). Thereby, carbon
polarization can be enhanced by up to a factor of four (γ
Η
/γ
C
), and advantage can be
taken from the much shorter T1 of protons (higher repetition rate).
The extend by which magnetization can be transferred to the heteronucleus by Hart
mannHahn cross polarization transfer depends on the mixing time, but also on the dis
tance between the nuclei. This technique may therefore be use to derive distance
information of pairs of nuclei:
1.5 Recoupling techniques in SSNMR:
Whereas magicangle spinning (MAS) leads to signiﬁcant linenarrowing in 1D spec
tra, the removed interactions need to be reintroduced in order to yield distance or
(dihedral) angle information. One way to yield information on spininteractions is to
spin the sample more slowly and extract spin interaction parameters from spinning
sideband amplitudes.
A more commonly technique used nowadays is to reintroduce spininteractions by
recoupling techniques. The basic idea behind recoupling techniques is that the averag
ing of spatialdependent parameter such as dipolar couplings or chemical shift aniso
tropy under MAS can be removed if RF pulses are applied in rotorsynchronized fash
1. Due to the fourfold higher γ of protons the energy difference between α and βstates is much larger
for protons and hence the population difference is also larger.
I
S
1.5
2.5
3.5
Mixing time [ms]
3 6
Distance in Å
Fig. Left: Pulse sequence for hetero CP. Right: Signal buildup curves for various dura
tions of mixing time and distances. A two spin system has been used and interactions
other than dipolar couplings are neglected.
LECTURE COURSE: NMR SPECTROSCOPY
Eleventh Chapter: SolidState NMR spectroscopy Pg.138
ion:
For heteronuclear dipolar recoupling rotational echo double resonance (REDOR) has
been used. Herein,
15
N,
13
C dipolar couplings are reintroduced during the mixing time
leading to dephasing of the signal by the dipolar coupling. Note that during signal
acquisition the dipolar couplings are removed by MAS so that narrow signals are sam
pled:
ω
r
ω
r
1/ 2ω
r
1/ 2ω
r
( )
n
0
0
S
H
IS
H
IS
S=
0 1 2 3 4 5 6 7 8
1
H
13
C
15
N
0 1 2 3 4 5 6
0
0.2
0.4
0.6
0.8
1
dephasing period (ms)
decoupling
t/τ
r
∆S
S
0

a)
b)
π/2
Fig.: a) REDOR pulse sequence for recording
13
C,
15
N dipolar couplings. b) Relative signal intensity
change against mixing time for a onebond dipolar coupling (from ref. Williamson et al.)
CP
CP
LECTURE COURSE: NMR SPECTROSCOPY
Eleventh Chapter: SolidState NMR spectroscopy Pg.139
Usually, a reference spectrum without the rotorsynchronized
15
N pulses is additionally
acquired and the intensities in both spectra are compared. The curves reﬂecting signal
intensity versus time depend on the magnitude of the heteronuclear dipolar couplings,
which in turn depend on their distance, and precise values for these can be extracted
from the data by comparison of the experimental data with simulations. Note that
strictly speaking the RF pulses are applied to the spinpart of the dipolar Hamiltonian
(I
z
S
z
) whereas MAS only inﬂuences the spatial part (3cos
2
θ1).
Homonuclear recoupling techniques have also been developed. Whereas its implemen
tation is similar to the heteronuclear variants in principle, it is complicated by the fact
that the spininteractions need to be reintroduced selectively. (A transformation I
z
S
z
>
I
z
(S
z
) is easily accomplished for a heteronuclear pair of spin by just applying a 180°
pulse to the S spin but usually difﬁcult for homonuclear spin pairs which will both be
ﬂipped unless the 180° pulse is selective). Selectivity in the homonuclear recoupling
techniques is achieved by adjusting a chosen property such as the spinning (rotor) fre
quency to the zero or doublequantum frequencies of the spin pair of interest (ω
zq
= ω
a

ω
b
, ω
dq
=ω
a
plus ω
b
). For example, in rotational resonance (R
2
) the rotor frequency is
set to a submultiple of the chemical shift difference. The dipolar coupling is then mea
sured in form of an exchange experiment: A resonance A is selectively inverted and the
exchange between the two sites is monitored:
Cross
Polarization
DANTE
Inversion
τ
m
π/2 π/2
π/2
Decoupling
a) b)
c)
δ (ppm)
70 60 50 40 30 20 10
ω
r
= 3411Hz
0 10 20 30
τ
m
(ms)
0.0
0.2
0.4
0.6
0.8
1.0
<
I
1
z

I
2
z
>
0ms
5ms
10ms
15ms
20ms
25ms
Fig.: a) Pulse sequence for a rotational resonance experiment. b) 1D spectra taken at various set
ting of the mixing time τ
m
. Note the change in signal amplitude of the signal at 53 ppm after the
resonance at 20 ppm has been selectively inverted. c) Change in intensity vs. mixing time.
CP
CP
LECTURE COURSE: NMR SPECTROSCOPY
Eleventh Chapter: SolidState NMR spectroscopy Pg.140
The condition for rotational resonance is fulﬁlled only for one (or at least a very few)
spin pairs.
Other techniques use double quantum transitions (HORROR, DREAM, DRAMA etc.),
and largely utilize rotorsynchronized RF pulses.
Jcouplings are much smaller and transfer times are much longer. In addition, all other
interactions need to be sufﬁciently eliminated. Therefore, methods relying on scalar
couplings are rather new and their use has been motivated by the fact that they allow to
trace along covalent networks (spin systems) leaving less ambiguity in the assignment
process.
1.6 SSNMR of oriented samples:
SSNMR spectra are complicated by the fact that
all kind of orientations with respect to the external
ﬁeld are possible. Some classes of molecules, e.g.
crystalline materials or peptides/proteins embedded
in phospholipid membranes, however, can be ori
ented either by deposition on glass plates or by
magnetic alignment. Assuming a unique associa
tion with the membrane the nuclei therefore do not
adopt all possible orientations with respect to the
external ﬁeld. This effect is demonstrated in the ﬁg
ure displayed on the left: The spectra were
recorded on
15
N fd coat protein on unoriented lipid
bilayers (C), magneticallyoriented lipid bicelles (A), and lipid bilayers oriented on
glass plates (B) (taken from ref. Opella et al.).
It has recently become clear that the orientation of helices with respect to the mem
brane normal may be probed from a combination of chemical shift and the onebond
dipolar
15
N,
1
H couplings in the socalled PISEMA spectra:
LECTURE COURSE: NMR SPECTROSCOPY
Eleventh Chapter: SolidState NMR spectroscopy Pg.141
.
The ﬁgure above displays the chemical shift of a
15
N amide resonance in dependence of
the orientation of the helix. In addition, the orientation of axes in the PAS are depicted.
Peaks belonging to amide moieties from a single helix in PISEMA spectra occur at
characteristic places, and the exact position and pattern can be rather easily related to
the angle the helix makes with the membrane surface (and hence with the ﬁeld):
In the ﬁgure above, A) displays peak position for various angles the helix makes with
Β
ο
220 140
σ{15Ν}
C
O
C
C
H
σ
11
σ
22
σ
33
Ν
α−1
α
θ=17
ο
A
C
B
β
LECTURE COURSE: NMR SPECTROSCOPY
Eleventh Chapter: SolidState NMR spectroscopy Pg.142
the membranenormal. In B) experimental peak positions are assigned to the helical
wheel positions for
15
N labeled AchR M2 peptide (a channelforming peptide) in ori
ented bilayers and compared to a simulated PISEMA spectrum calculated for a tilt
angle of 12°. In C) typical PISEMA spectra are displayed for the AchR M2 peptide
(right) together with the HSQC spectrum recorded in solution on the DPCmicelle
bound form.
1.7 Labeling strategies for solidstate NMR applications:
The easiest (and most often used) remedy to reduce the vast number of interactions in
the solid state is the use of selectively labeled material, in which NMR active isotopes
(
13
C,
15
N) are only introduced at speciﬁc sites.
Whereas selectively labeled small molecules usually need to be synthesized from suit
able precursors, peptides can be subsequently labeled at cysteine sites with methylio
dide. Alternatively, biosynthetic routes to prepare alternatingly labeled peptides (only
every second position is labeled) or selectively at methyl position have been developed.
Assigning polypeptides from uniformly labeled material still presents a major chal
lenge. For that purpose a series of tripleresonance experiments relying on transfer
pathways similar to those used for solution studies have been suggested.
Another common problem is that signals are inherently broadened due to the structural
disordered in noncrystalline solids. Hence, microcystalline solids are usually required
and much attention needs to be paid to sample preparation.
References:
• P.T.F. Williamson, M. Ernst, B.H. Meier: MAS SolidState NMR of
Isotropically Enriched Biological Samples in BioNMR in Drug Research, Ed.
O. Zerbe, Wiley 2003.
• R. Tycko, Biomolecular Solid State NMR: Advances in Structural Methodology
and Applications to Peptide and Protein Fibrils, Annu. Rev. Phys. Chem. (2001),
52, 575606.
• D.D. Laws, H.M. Bitter, A. Jerschow, SolidState Spectroscopic Methods in
Chemistry, Angew. Chem. (2002), 41, 30963129.
• S.J. Opella, C. Ma, F.M. Marassi, Nuclear Magnetic Resonance of Membrane
Associated Peptides and Proteins, Meth. Enzymol. (2001), 339, 285313.
LECTURE COURSE: NMR SPECTROSCOPY
Scalar couplings: 60 Direct couplings (1J): 62 Geminal couplings (2J): 63 Vicinal couplings (3J): 63 Longrange couplings: 65 Couplings involving p electrons: 65 The number of lines due to scalar spin,spin couplings: 65 Strong coupling: 67 68 T1 relaxation: 68 T2 relaxation: 69 The mechanisms of relaxation: 70 Other relaxation mechanisms: 72 Chemical shift anisotropy (CSA): 72 Scalar relaxation: 72 Quadrupolar relaxation: 73 Spinrotation relaxation: 73 Interaction with unpaired electrons: 73 The motional properties: 73 The dependence of the relaxation rates on the fluctuating fields in x,y or z direction: 75 Excurs: The LipariSzabo model for motions: 77 The nature of the transitions: 78 Measurement of relaxation times: 80
Relaxation:
The Nuclear Overhauser Effect (NOE): 84 Experiments to measure NOEs: 86 The steadystate NOE: 87 Extreme narrowing (hmax >0): 87 Spindiffusion (hmax <0): 88 The transient NOE: 89 The state of the spin system and the density matrix: 90 The sign of the NOE: 92 Why only zero and doublequantum transitions contribute to the NOE 94 Practical tips for NOE measurements: 96 Chemical or conformational exchange: Twosite exchange: Fast exchange: The slow exchange limit: The intermediate case: Investigation of exchange processes: EXSY spectroscopy: Saturation transfer: Determination of activation parameters: 99 99 101 102 102 103 103 104 105
2
LECTURE COURSE: NMR SPECTROSCOPY
The product operator formalism (POF) for description of pulseexperiments: 106 RF pulses: 107 Chemical shift precession: 107 Scalar spin,spin coupling: 108 A simple onedimensional NMR experiment: 110 The effect of 180 degree pulses: 111 Coherence transfer: 112 Polarization transfer: 113 TwoDimensional NMR Spectroscopy: The preparation period: The evolution period: The mixing period: The detection period: Hetcor and inversedetection experiments: Phasecycling: An Alternative: Pulsed Field Gradients Hybrid 2D techniques: Overview of 2D experiments: Original references for 2D experiments: Solid State NMR Spectroscopy: The chemical shift Dipolar couplings: Magic Angle Spinning (MAS) Sensitivity Enhancement: Recoupling techniques in SSNMR: SSNMR of oriented samples: Labeling strategies for solidstate NMR applications: 118 120 121 122 124 124 125 126 128 129 130 132 132 135 136 136 137 140 142
3
liquidstate) determination of the dynamics in the solidstate Imaging techniques spin tomography "In vivo" NMR spectroscopy distribution of metabolites in the body
4
. determination of optical purity Biopolymers up to about 2030 kDa determination of the 3D solution structure provided the primary sequence is known! investigation of the interaction of molecules (complexes) investigation of the dynamics of proteins
1H.trans isomerism.1H
or 15N relaxation measurements
oder 1H. 13C 1H.15N NOE measurements
Determination of the kinetics of reactions
Solidstate NMR spectroscopy insoluble compounds (synthetic polymers) very large compounds (requires speciﬁc labels) determination of the structure in the solidstate (vs.LECTURE COURSE: NMR SPECTROSCOPY
APPLICATION FIELDS OF NMR SPECTROSCOPY
Highresolution NMR spectroscopy Analytics "small" molecules determination of the covalent structure determination of the purity Elucidation of the 3D structure "small" molecules determination of the stereochemistry: cis.
The faster the charge travels the stronger is the induced magnetic ﬁeld. In other words. THE PHYSICAL BASIS OF THE NMR EXPERIMENT
Imagine a charge travelling circularily about an axis. are expected to align with the direction of the magnetic ﬁeld. when placed into a magnetic ﬁeld. a magnetic dipole has been created. The torque that acts to rotate the needle may be described as ∂J = r×F ∂t in which J is deﬁned as the angular momentum which is the equivalent for rotaT =
First Chapter: Physical Basis of the NMR Experiment Pg.5
. This is similar to a current that ﬂows through a conducting loop: µ
FIGURE 1.
When such a compass needle is turned away from the northpole pointing direction to make an angle φ a force acts on the needle to bring it back.
Such a circular current builds up a magnetic moment µ whose direction is perpendicular to the plane of the conducting loop.LECTURE COURSE: NMR SPECTROSCOPY
1. For the case of a dipole moment that has been created by a rotating charge this force is proportional to the strength of the ﬁeld (B) and to the charge (m). Such dipoles. In the following we will look at a mechanical equivalent represented by a compass needle that aligns within the gravitational ﬁeld:
N
φ
FIGURE 2.
The torque is formed by the vector product between the radius and the momentum (see additional material) and is described by a vector which is perpendicular to both radius and momentum. Rotation of the nuclear momentum about its own axis (blue) and about the magnetic ﬁeld axis (red). it is the axis of rotation which is perpendicular to the plane. In fact.6
.
First Chapter: Physical Basis of the NMR Experiment
Pg.LECTURE COURSE: NMR SPECTROSCOPY
tional movements of the linear momentum.
J=rxp p=mv FIGURE 3. The corresponding potential energy is
ϕ
E pot = – ∫ T dϕ
0
In contrast to the behaviour of a compass needle the nuclear spin does not exactly align with the axis of the external ﬁeld:
ω B
FIGURE 4. Left: linear momentum.) Inertia Force (Torque) Momentum Work Kinetic energy Power x v = dx/dt a = dv/dt m F p = mv W = Int F dx K = 1/2 mv P=Fv
2
Pure Rotation (fixed axis) θ ω =dθ/dt α = dω/dt I T=r xF J=rxp W = Int T dθ K = 1/2 Iω2 P=Τω
Note that the direction of the momentum is tangential to the direction along which the particle moves. Right: angular momentum
Excurse: Corresponding parameter for translational and rotational movements
PureTranslation (fixed direction) Position Velocity Acceleration Translational (Rot.
the gyroscope precesses about the axis perpendicular to the ground with a frequency ω that is called the precession frequency (right side) and takes a welldeﬁned angle φ with respect to the rotation axis: ω
φ
The frequency ω is expressed in terms of a angular velocity (see additional material). Hence. we do not observe single molecules but look at an ensemble of molecules (usually a huge number of identical spins belonging to different molecules). below. When the this gyroscope is placed with the shaft perpendicular to the ground and released it will fall down (see Fig. It rotates (spins) about its own axis (the blue arrow) and precesses about the axis of the magnetic ﬁeld B (the red arrow).LECTURE COURSE: NMR SPECTROSCOPY
This is a consequence of its rotation about its own axis. This property is called spin.13C. The frequency of the precession is proportional to the strength of the magnetic ﬁeld: ω=γB The proportionality constant is called the gyromagnetic ratio.
Excurse: The movement of a classical gyroscope Imagine a wheel ﬁxed to a shaft. The precession frequency
ω0 = ν ο 2 π
is called the Lamor frequency. The sum of the dipole moments of identical spins is called magnetization: M =
∑ µi
j
Pg.7
First Chapter: Physical Basis of the NMR Experiment
. In contrast to a compass needle which behaves "classically" in the way that it can adopt a continous band of energies depending only on the angle φ it makes with the ﬁeld the corresponding angle φ of the nuclear dipole moment is quantitized. left side). It is speciﬁc for the kind of nucleus and therefore has a different value for 1H. we will later introduce the quantummechanical treatment shortly. Of course. when the wheel spins about the axis of the shaft.19F etc. However.
1 The Bloch equations: The Bloch’s equations describe the fate of magnetization in a magnetic ﬁeld.8
. In the absence of an additional B1 ﬁeld the components of the ﬁeld along the cartesian axes are: Bx = 0 By = 0 Bz = Bo which leads to the following set of coupled differential equations: ∂ M ( t ) = γ My B0 – Mx ⁄ T2 ∂t x ∂ M ( t ) = –γ Mx B0 – My ⁄ T2 ∂t y ∂ M ( t ) = –( Mz –M0 ) ⁄ T1 ∂t z In order to drive the system into the equilibrium state (no transverse coherence.
First Chapter: Physical Basis of the NMR Experiment Pg. material for the mathematics involved): T Considering that (see page 2) T = and that M = ∑ µi = ∑ γ Ji i i ∂J ∂t
=
M×B
we ﬁnd that ∂ ∂M = γ J = γT = γ ( M × B ) ∂t ∂t which describes the timeevolution of the magnetization. Mathematically this is decribed by forming the vector product between dipole moment and magnetic ﬁeld (see add.LECTURE COURSE: NMR SPECTROSCOPY
1. relative population of the α/ β states according to the Boltzmann distribution) additional terms have been phenomenologically introduced such as Mx/T2 for the Mx component. We have stated before that a force ( a torque) acts on a dipole moment when it is placed inside a mgnetic ﬁeld such that the dipole moment will be aligned with the direction of the static magnetic ﬁeld.
. I+1. Vectorial representation of the zcomponent of the angular momentum for a spin I=1/2 (left) and spin I=1 (right) nucleus. the zcomponent of the angular momentum J is quantitized and can only take discrete values
Jz = m
h 2π
where m is the magnetic quantum number. For spins with I=1 nuclei three different values for Jz are allowed:
First Chapter: Physical Basis of the NMR Experiment
Pg. Therein. The latter can adopt values of m = I. When looking at single spins we have to use a quantummechanical treatment. giving rise to two distinct energy levels.
Hz Hz
Jz = + Jz = 0 Jz= 
I=1/2
I=1
Jz= +
h 4π h 4π
h 4π
Jz = 
h 4π
FIGURE 5..
For I=1/2 nuclei.0. …...LECTURE COURSE: NMR SPECTROSCOPY
The solutions to these equations are given by:
M x ( t ) = [ M x ( 0 ) cos ωt – M y ( 0 ) sin ωt ]e M z ( t ) = M eq + [ M z ( 0 ) – M eq ] e
( – t ⁄ T2 ) ( – t ⁄ T2 )
M y ( t ) = [ M x ( 0 ) sin ωt + M y ( 0 ) cos ω t ]e
( – t ⁄ ( T1 ) )
The ﬁrst two equations describe mathematically a vector that precesses in a plane (see add. 1. m can only be +1/2 or 1/2.9
.2 Quantummechanical treatment: The dipole moment µ of the nucleus is described in quantummechanical terms as
µ= γ J
Therein. J is the spin angular momentum and γ the gyromagnetic ratio of the spin. mat.) and hence give the correct description for what we will be looking at in a rather pictorial way in the following.I1. I with I being the spin quantum number.
Energy levels of the α.and β.and βstates (left). In equilibrium the spins of I=1/2 nuclei are either in the α.
First Chapter: Physical Basis of the NMR Experiment
Pg.states of I=1/2 nuclei
1. their phases are not correlated. that only transitions with ∆m=
Jz= +
m= +1/2
β
+ 1 / 2 γ h / 2π B
∆ E=hν
Jz= .1 / 2 γ h / 2π B
FIGURE 6. we now look at the macroscopic bevaviour.h
4π
m= 1/2
α
Bo = 0 Bo ≠ 0
.3 The macroscopic view: The NMR experiment measures a large ensemble of spins derived from a huge number of molecules.LECTURE COURSE: NMR SPECTROSCOPY
E pot =
2.10
. Equilibrium state with similarily populated α. which corresponds to the two states with m=±1/
E pot = γ
±1 are allowed):
Hz h 4π
Ε
(the quantummechanical selection rule states. Therefore. However. is then
µzB = γ J B
h B 2π
The energy difference ∆Epot. uncorrelated phases (middle) and no net phase coherence (right). For each vector pointing in one direction of the transverse plane a corresponding vector can be found which points into the opposite direction:
Bo=z Bo=z
y x x y y
ΣΣ
y x x y y x x
FIGURE 7. The sum of the dipole moments of all nuclei is called magnetization.or βstate and precess about the axis of the static magnetic ﬁeld.
the socalled B1 ﬁeld. This state is called coherence. application of a radiofrequency (RF) ﬁeld perpendicular to the magnetic ﬁeld (e. Bo=z y x
y x y x
Σ
FIGURE 8. Therefore. only the vector sum of the transverse component is
First Chapter: Physical Basis of the NMR Experiment
Pg.LECTURE COURSE: NMR SPECTROSCOPY
Therefore. Coherent state of spins in α. creates a state in which the phases of the spins are partially correlated.y plane) is observed.y plane (the vector sum of the transverse components) is vanishing provided that the phases of the spins are uncorrelated.or yaxis).or β(left) states and the projection onto the x.y plane the resulting transverse magnetization is nonvanishing giving rise to a signal in the detection coil. along the x.11
.g. Only transverse magnetization leads to a detectable signal in the receiver coil and hence contributes to the NMR signal. the projection of all vectors onto the x. When the motions of spins are described in vector diagrams most frequently only the vector sum of the spins is shown in order to simplify it. This is shown in the following ﬁgure in which the vector sum of longitudinal (blue) and transverse (yellow) magnetization of uncorrelated spins which are only in the αstate (A) or in both states (B) is displayed as well as for correlated states (C and D): It is evident that only for correlated states transverse magnetization (magnetization in the x.y plane(middle) and sum vector of the x.
The magnitude and direction of the magnetization vector can be calculated by vectorial addition of the separate dipole moments.y component. When projecting the vectors onto the x.
However.
LECTURE COURSE: NMR SPECTROSCOPY
shown to describe the relevant part of the spins:
A
Bo=z Bo=z
B
Bo=z Bo=z
y x x
y x
y
y x
C
Bo=z y x Bo=z
D
Bo=z y x Bo=z
y x
y x
FIGURE 9. Different states of the spin system (see text).
The experiment setup of the spectrometer includes a radiofrequency coil. which delivers the orthogonal B1 ﬁeld.12
.
The magnitude of the current that is induced in the receiving coil depends on the orientation of the magnetization vector with respect to the coil.y) plane induces the NMR signal it is convenient to look at the vector sum of the transverse components which present a magnetic ﬁeld that rotates in space:
z
x y
z
x
z
x
∆t
y
2∆t
y
FIGURE 10. To understand how the magnetization that rotates in the transverse (x. Simultaneously this coil serves to pick up the NMR signal. Spins precessing at different velocities (and hence have different chemical shifts) are color coded. When the
First Chapter: Physical Basis of the NMR Experiment
Pg.
The amplitude of the induced current is following a (co) sine wave. Spins with different chemical shift. Similarily.LECTURE COURSE: NMR SPECTROSCOPY
magnetization is pointing towards the coild the induced current is at maximum. e. Because the magnetization rotates the induced current follows a sine (or cosine) wave (see additional material). Left: Conducting loop rotating in a magnetic ﬁeld with corresponding current induced (right).
In the picture above the induced current is shown for different orientations of the transverse magnetization. This situtation is very similar to a conducting loop that rotates in a magnetic ﬁeld as encountered in a generator:
I
N
S
t
FIGURE 12. different larmor frequencies.
First Chapter: Physical Basis of the NMR Experiment
Pg. Left:Rotating spin with its position at certain time intervals 16 are marked. Right: Corresponding signal in the receiver coil. opposite to the situation in a NMR experiment. precess at different rates and hence the frequency of the current is the larmor frequency. the frequency of the precessing spins: .g.
However. the rotating dipoles in NMR create a sinemodulated current.
z
5 4 2 x 6 1 y 3
I Det
1 2 6 3 5
t
4
T
FIGURE 11.13
. in the generator the (coil) loop is rotating and the magetic ﬁeld is stationary.
For more complex compounds that contain many different spins the signal in the receiver coil is a superposition of many different frequencies. To emphazise that this ﬁeld is turned on for only a very short period of time usually it is called a (RF) pulse. This phenomenon is called relaxation and leads to decreasing amplitude of signal in the detector with time. the magnetization does not precess in the transverse plane for inﬁnite times but returns to the zaxis. To create spincoherence an additional RF ﬁeld is switched on. 1.
First Chapter: Physical Basis of the NMR Experiment
Pg.4 Fourier Transform NMR: Spins that belong to nuclei with different chemical environment precess with different frequencies. During the time where B0 and B1 ﬁeld are both present the magnetization rotates about the axis of the resulting effective ﬁeld. The Fourier Transformation is a convenient mathematical tool for simultaneous extraction of all frequency components. Signals from 3 spins with different precession frequencies (left) and their corresponding Fourier transforms (right)
In reality.14
.LECTURE COURSE: NMR SPECTROSCOPY
1. that is perpendicular to the axis of the static ﬁeld (the socalled B1 ﬁeld).5 The interaction between the magnetization and the additonal RF (B1) ﬁeld: When only the static B0 ﬁeld is present the spins precess about the zAxis (the axis of the B0 ﬁeld). It allows to transform data from the time into the frequency domain:
I(t)
→
FT
I( ν )
I(det)
t Α Β
FT
t
C
ν
C
Β
Α
FIGURE 13.
the B0 ﬁeld corresponds to precession frequencies of hundreds of MHz whereas the B1 ﬁeld is about 120 KHz). If the B1 ﬁeld would be applied ﬁxed along the xaxis all the time it would have an negligible inﬂuence onto the spins.LECTURE COURSE: NMR SPECTROSCOPY
This effective ﬁeld is calculated by taking the vector sum of the B0 and B1 ﬁeld: ω0 Bo Bo
Bo Beff
B1
ω1 B1
FIGURE 14. Movement of spins in the presence of only the B0 ﬁeld (left). left: Static Bo ﬁeld and rotating B1 ﬁeld.g.15
. middle: Laboratory frame frequencies.
For those spins whose lamor frequency is exactly ωο the effective Bo ﬁeld they
First Chapter: Physical Basis of the NMR Experiment Pg. To understand the effect of the rotating B1 ﬁeld. Bo and B1 ﬁeld (middle) and vector additon to calculate the effective ﬁeld formed by Bo and B1
Now. a frame in which the coordinate system is not ﬁxed but rotates with ωο):
Bo ω ω0 y x B1
ω − ω0 ω0
Bo
ω
Bo
ω
FIGURE 15. it is very convenient to transform into a coordinate system that rotates with the precession frequency ωo of the B0 ﬁeld (in the picture above that means that the small "blue" man jumps onto a platform that rotates with the larmor frequency of the spin. However. right: Rotating frame frequencies. the B1 ﬁeld is not static but rotates about the zaxis (the axis of the static ﬁeld) with a frequency that is very similar to the precession frequency of the spins about the zaxis. This operation transforms from the laboratory frame to a rotating frame. the B0 ﬁeld is larger than the B1 ﬁeld by many orders of magnitude (for protons. e.
LECTURE COURSE: NMR SPECTROSCOPY
experience in such a rotating reference frame is zero. These spins then only feel the effect of the B1 ﬁeld and precess about the B1 axis as long as the B1 ﬁeld is turned on. Spins that have a slightly different precession frequency ω the strength of the B0 ﬁeld is B’=B0ω/γ (For ω=ω0, B’ is zero). During RF pulses spins which are exactly onresonance (e.g. whose precession frequency is equal to the precession frequency of the B1 ﬁeld about the zaxis) only feel the B1 ﬁeld. These spins precess about the axis of the B1 ﬁeld until the B1 ﬁeld is turned off again:
B 0  ω/γ
B’
Beff
Beff
B’
Beff B1
Beff
B1
B1
ωo
ωo
ωo
FIGURE 16. 1st : Vectorial addition of B1 and Bo ﬁeld. 2nd: Direction of the effective ﬁeld for spins exactly onresonance, 3rd more offresonance, 4th far offresonance.
The overall ﬂipangle they have experienced during that time tp is α = γ B1 tp Usually the pulse lengths tp are choosen such that the ﬂip angle α is 90 or 180 degrees. As stated above the effect of the B1 ﬁeld is to create phase coherence amongst the spins. The macroscopic effect of coherent spins is transverse magnetization. 1.6 Description of the effect of the B1 ﬁeld on transverse and longitudinal magnetization using the Bloch equations: We have seen before that ∂M = γ (M × B) ∂t Now, Bx = B1cosω0t
First Chapter: Physical Basis of the NMR Experiment Pg.16
LECTURE COURSE: NMR SPECTROSCOPY
By = B1sinω0t Bz = Bo and hence ∂ M ( t ) = γ ( M y B 0 + M z B 1 sin ω 0 t ) – ( M x ⁄ T 2 ) ∂t x ∂ M ( t ) = – γ ( M x B 0 – M z B 1 cos ω 0 t ) – ( M y ⁄ T 2 ) ∂t y ∂ M ( t ) = – γ ( M x B 1 sin ω 0 t + M y B 1 cos ω 0 t ) – ( M z – M 0 ) ⁄ T 1 ∂t z To solve these equations they are conveniently transformed into a reference frame that rotates at the frequency ω0 about the zaxis. Therein M x = M x cos ω 0 t – M y sin ω 0 t M y = M x sin ω 0 t + M y cos ω 0 t In this "primed" coordinate system the equations become: ∂M x ′ ′ = ( ω 0 – ω )M y – ( M x ⁄ T 2 ) ∂t ′ ∂M y ′ ′ = –( ω0 – ω ) Mx + γ B1 Mz –My ⁄ T2 ∂t ′ ∂M z ′ = –γ B1 My – ( Mz – M0 ) ⁄ T1 ∂t The solutions are rather involved and will not presented here since the signals are usually recorded when only the B0 ﬁeld is present nowadays. However, it is important to see that in this frame the effect of the B1 ﬁeld on transverse magnetization aligned along the y' axis is to rotate it about x' with a frequency of ω1= γ B1.
′ ′ ′
1.7 The excitation proﬁle of pulses: A pulse is a polychromatic source of radiofrequency and it covers a broad band of frequencies. The covered bandwidth is proportional to the inverse of the pulse duration. Short pulses are required for uniform excitation of large bandwidths, long (soft) pulses lead to selective excitation. Usually the nonselective (hard) pulses have B1 ﬁelds of the order of 520 kHz and pulselengths of 520 µs, whereas selective (soft) pulses may last for 1100 ms with an appropriately
First Chapter: Physical Basis of the NMR Experiment Pg.17
LECTURE COURSE: NMR SPECTROSCOPY
attenuated RF amplitude:
FIGURE 17. Left: Excitation proﬁle of a “hard” pulse, right: Ex. proﬁle of a “soft” (long) pulse
1.8 Relaxation: The magnetization does not precess inﬁnitely in the transverse plane but turnes back to the equilibrium state. This process is called relaxation. Two different timeconstants describe this behaviour: a) the reestablishment of the equiilibirum α/β state distribution (T1) b) dephasing of the transverse component (destruction of the coherent state, T2). The T2 constant characterizes the exponential decay of the signal in the receiver coil:
FIGURE 18. Right: Signal in absence of transverse relaxation, right: real FID (free induction decay)
The precessing spins slowly return to the zaxis. Instead of moving circularily in the transverse plane they slowly follow a spiral trajectory until they have
First Chapter: Physical Basis of the NMR Experiment
Pg.18
right: individual x. The oscillation occurs periodically so that the displacement from the equilibrium position follows a cosine timedependence. left: Trajectory of the magnetization.y.zaxis:
FIGURE 19.z component
A mechanical equivalent is a spring oscillating in time. Monitoring the position of a weight ﬁxed to a spring depending on the time elapsed after release can be described by an damped oscillation. Because of frictional energy loss the oscillation is damped so that after some time the spring is not oscillating anymore:
FIGURE 20.LECTURE COURSE: NMR SPECTROSCOPY
reached their inital position aligned with the +/.19
. It charac
First Chapter: Physical Basis of the NMR Experiment
Pg.
The damping timeconstant is called T2 or transverse relaxation time.
The signal intensity is proportional to the population difference of the two states. The width of the signal at half height is given by: ∆ν 1 ⁄ 2 = 1 ⁄ ( πT 2 ) Fast decay leads to broad signals.LECTURE COURSE: NMR SPECTROSCOPY
terizes the time it takes so that the signal has decayed to 1/e of its original magnitudeThe transverse relaxation constant T2 is related to the linewidth of the signals.the overall tumbling time of the molecule which is related to its size. The relative population of the states can be calculated from the Boltzmann distribution:
(E β − E α ) kT
N = e Nβ
α
with T being the measuring temperature and k the Boltzmann constant.
The transverse relaxation constant T2 of spin I=1/2 nuclei is mainly governed by . depending on the number and the distance of neighbouring nuclei . rapidly decaying ones to broad lines (right). Slowly decaying FIDs lead to narrow lines (left).9 The intensity of the NMR signal: If the α and β states would be populated equally. 1. EβEα
First Chapter: Physical Basis of the NMR Experiment
Pg.the homogeneity of the magnetic ﬁeld ( the "shim") . no netto change in energy could be observed.the strength of the dipolar interaction with other I=1/2 nuclei. slow decay to sharper lines:
FIGURE 21.20
.
∆E also depends on Bo.98 1. [%] 99.11 100 0. the required quantities of sample are much larger.2712 1. The energy difference ∆E depends on the gyromagnetic ratio γ:
E=γ
nucleus
1H 13C 19F 15N 31P
h Bo 2π
Srel 1.083
Nat.76 3. and therefore the signal is much weaker. the natural abundance of 1H is 300 times larger than for 15N. Unfortunately. much smaller than in IR or UV spectroscopy.37 100
Since γ of
is approx.00 1. Abd.LECTURE COURSE: NMR SPECTROSCOPY
is the energy difference between the α.0665
15N
IN 1/2 1/2 1/2 1/2 1/2
1H
γ [108 T1s1] 2.517 0. Direct observation of 15N nuclei requires high concentrations and long measuring times. Higher ﬁelds lead to dramatically reduced measurement times. Furthermore.and the βstate.673 2. In other words. To be more precise the signal that is induced in the receiver coil depends on both the gyromagnetic ratio of the excited and detected spin: Int prop γex * γdet3/2
First Chapter: Physical Basis of the NMR Experiment
Pg.21
. 10 times larger for 1H than for
Nα/Nβ is much
larger and the signal much more intense.675 0.85 104 106 0.83 0. the energy difference in NMR experiments is very small.
Schematic drawing of the magnet. coils and probehead
Second Chapter: Practical Aspects
Pg. THE COMPONENTS OF A NMR INSTRUMENT
The NMR instrument consists mainly of the following parts: • • • • • • • • the magnet probehead(s) radiofrequency sources amplifiers analogdigital converters (ADC's) the locksystem the shim system a computer system
1.22
. the main coil that produces the ﬁeld is placed in a liquid helium bath so that the electric resistance of the coil wire is zero.LECTURE COURSE: NMR SPECTROSCOPY
1. N2
dewar
liq. He dewar
Main Coil
Probehead
FIGURE 1. showing He and N2 dewars. The helium dewar is surrounded by a liquid nitrogen dewar to reduce loss of the expensive helium:
roomtemperature shim tube
liq. Therein.1 The magnet system: Usually the static magnetic ﬁeld nowadays is provided by a superconducting (supercon) magnet.
The inner coil is more sensitive (higher S/N) and gives shorter pulselengths. 1.LECTURE COURSE: NMR SPECTROSCOPY
Additional socalled cryocoils are placed in the Hebath to correct partially for ﬁeld in homogeneity.2 The probehead: The probehead contains the receiver/transmitter coil (actually only a single coil for both purposes):
˜
˜
Spinner
Transmitter/ Receiver Coil
˜
˜
Resonance Circuit
Cables to Preamp
FIGURE 2. shim coils are located. The sample that is mostly contained in a deuterated solvent inside a 5mm glastube is placed inside a spinner and then lowered through the r.23
. An inverse carbon probe has the inner coil tuned to 2H and 1H and the outer coil to 13C. It has high proton sensiSecond Chapter: Practical Aspects Pg. The magnet is vertically intersected by the roomtemperature shim tube.t. These are the ones that have to be adjusted by the user. The deuterium channel is placed on most probes on the inner coil. most probes contain two coils: An inner coil tuned to deuterium (lock) and a second frequency and an outer coil tuned to a third and possibly a fourth frequency.t. View of the spinner with sample tube placed in probehead
However. shim tube so that it enters the probehead from the top. On its surface the r.
The frequency synthesizers nowadays are completely digital. The analogdigital converters (ADC's) are required because the signal is recorded
Second Chapter: Practical Aspects
Pg. The ampliﬁers are lownoise audioampliﬁers which boost the outgoing and incoming signals.1 Hz.24
. He System
Probehead
Preamp
FIGURE 3. The receiver/transmitter coil should not be confused with the cryocoils (coils placed in the He bath) that produce the static ﬁeld. Once the spinner is lowered the NMR tube is positioned such that the liquid inside is covered by the receiver coil completely.LECTURE COURSE: NMR SPECTROSCOPY
tivity and is wellsuited for inverse detection experiments like HSQC or HMBC. They are in the range of 50100 Watts for protons and 250500 Watts for heteronuclei. The resonance circuit inside the probe also contains the capacitors that have to be tuned when changing the sample (especially when the solvent is changed). Magnet and preamplifier system
The radiofrequency sources (frequency synthesizers) are electronic components that produce sine/cosine waves at the appropriate frequencies. Part of them are often modulaters and phase shifters that change the shape and the phase of the signals. The preampliﬁer also contains a wobbling unit required for tuning and matching the probe:
liq. N 2 Inlet
liq. The probehead is then connected to the preampliﬁer which is usually placed close to the magnet and which performs a ﬁrst ampliﬁcation step of the signal. A carbon dual probe has the inner coil tuned to 2H and 13C and the outer to 1H and is dedicated for carbon experiments with proton decoupling.Frequency resolution is usually better than 0. The receiver coil is in the center of the magnetic ﬁeld.
LECTURE COURSE: NMR SPECTROSCOPY
in analog form (superposition of various damped harmonic oscillations) but must be in digital form in order to be accessible to Fourier transformation by the computer. In modern nmr spectrometers 16 or 18 bit digitizers are used. This means that the most intensive signal can be digitized as 218. This has severe consequences for the dynamic range because it implies that signals with less than 1/218 of the signal amplitude cannot be distinguished in intensity. Therefore, the receiver gain (the ampliﬁcation of the signal) should be adjusted such that the most intense signal almost completely ﬁlls the ampliﬁer. However, some care has to be paid in experiments that utilize water suppression: Sometimes the most intense signal in a 2D experiment does not occur in the ﬁrst increment because the water may be (due to radiation damping) stronger in later increments. It is advisable to set the evolution time to longer values (e.g. 100ms) and observe the intensity of the water signal. The computer system is an industrystandard UNIX machine with lots of RAM. However, offside processing on PCs is also possible and the PC is also started being used as host computers on the spectrometer (unfortunately). 1.3 The shim system: Considering that the precession frequencies are proportional to the magnetic ﬁeld strength the magnetic ﬁeld has to be highly homogenous across the sample volume in order to be able to observe small frequency differences (small couplings). If the ﬁeld would not be highly homogenous, the effective ﬁeld strengths in different volume compartments inside the sample would be different and the spins therefore would precess at different rates. This would lead to considerable linebroadening (inhomogeneous broadening). The shimsystem is a device that corrects for locally slightly different magnetic ﬁelds. The shimsystem consists of two parts: (A) the cryoshimsystem and (B) the roomtemperature shims. The basic principle behind them is the same. Small coils are supplied with regulated currents. These currents produce small additional magnetic ﬁelds which are used to correct the inhomogeneous ﬁeld created by the main coil. There are a number of such coils of varying geometry producing correction ﬁelds in different orientations. In the cryoshim system the coils are placed in the He bath, they are usually only adjusted by engineers during the initial setup of the instrument. The roomtemperature shim system is regulated by the user whenever a new sample is placed in the magnet or when the temperature is changed. The room temperature shim system is grouped into two sets of shims • • the onaxis (spinning) shims (z,z2,z3,z4..) the offaxis (nonspinning) shims (x,y,xy...)
Second Chapter: Practical Aspects
Pg.25
LECTURE COURSE: NMR SPECTROSCOPY
The onaxis shims only correct for inhomogeneity along the zaxis. They are highly dependent on the solvent and the solvent ﬁlling height. At least the lower order gradients (z, z2 and z3) should always be adjusted. The z4 gradient depends very much on the ﬁlling height and its adjustment is tedious. Therefore, in order to speed up the shimming process, the ﬁlling height should (if possible) always be the same (e.g. 500 or 550 µl) and a shimset for that height and probehead should be recalled from disk. The offaxis shims usually do not have to be adjusted extensively (only x, y, xz and yz routineously). Wrong offaxis shims lead to spinning sidebands when the sample is rotated during measurement. Their contribution to the lineshape is largely removed with spinning but this unfortunately introduces disturbances. It should therefore be avoided during 2D and NOE measurements. The name of the onaxis shims is derived from the order of the polynomial that needs to be used to correct for the ﬁeld gradient along the zaxis:
Bo
z
z2
z3
z4
FIGURE 4. Field dependence of onaxis shims
The zshim delivers an additional ﬁeld that linearly varies along the sample tube. The z2 shim has its largest corrections to the ﬁeld at the top and the bottom of the sample.
For the more experienced spectroscopist the misadjusted onaxis shim can mostly be recognized from the signal lineshape. Misadjusted shims with even exponentials (z2,z4,z6) give asymmetric signals, those with odd exponentials (z1,z3,z5) show up as a symmetrical broadening. This is due to the symmetry of the function needed to correct of it. Z4 for example increses the field strength at the the top and the bottom of the sample and hence gives only frequencies larger than the correct frequency. Z3 in contrast increases the freuqnecy at the top and decreases the frequency at the bottom of the sample. Therefore, in the case of z4, there is a proportion of singal shifted to higher frequencies and hence to one side of the signal, whereas z3 give proportions of the signals shift to lower and higher frequencies resulting in asymmetric and symmetric errors for the former and latter, respectively. The closer the bump is to the base of the signal the higher the gradients needs to be to correct for it.
Shimming is usually performed by either observing the intensity of the lock
Second Chapter: Practical Aspects
Pg.26
LECTURE COURSE: NMR SPECTROSCOPY
signal (vide infra) or by monitoring the shape of the FID.:
FIGURE 5. Misadjusted shims and appearance of corresponding signals after FT.
Another way of controlling the homogeneity of the magnetic ﬁeld is to watch the shape of the FID. When the ﬁeld is highly homogenous, the FID should fall smoothly following an exponential. The resolution of the signal determines how long the FID lasts:
FIGURE 6. FID of perfectly shimmed magnet (left) and misshimmed magnet (right).
Second Chapter: Practical Aspects
Pg.27
that means distinguish frequencies of spins rotating slower than the carrier frequency from those that precess at higher rates:
a) single detector
ν
0
+ν
ν
0
+ν
FIGURE 7. the received signal is also transformed down to audio frequency.LECTURE COURSE: NMR SPECTROSCOPY
1.
Second Chapter: Practical Aspects
Pg. that coincides with the center frequency of the recorded spectrum.Thereby.4 The locksystem: Stability of the magnetic ﬁeld is achieved by the deuterium lock system. which generates corrections to the magnetic ﬁeld strength Bo. We have seen before that the B1 ﬁeld rotates about the axis of the static ﬁeld with a certain frequency. mostly 2D measurements and even more importantly. Consequently.28
. To provide good sensitivity of the lock the lock power should be adjusted just below saturation (lockline must still be stable!). The system has a feedback loop. The stability is inﬂuenced by many factors of which the temperature instability has the largest inﬂuence. Similarly. In addition we require quadrature detection: A single coil cannot distinguish positive and negative frequencies. Top: Signal due to negative frequency. Hence. such that the resonance frequency of the solvent deuterium line remains constant. When the locksystem is not activated the naturally occurring drift of the magnetic ﬁeld leads to varying resonance frequencies over time and hence to linebroadening. The deuterium lock measures the frequency of the deuterium line of the solvent. Modern digital lock system allow to adjust the regulation parameters of the lock channel.5 The transmitter/receiver system: It is much more convenient to handle audiofrequencies than handling radiofrequencies. deuterated solvents have to be used for FTNMR. For experiments utilizing pulsedﬁled gradients the lock should be highly damped in order to avoid that the lock tries to correct each time the ﬁeld recovers from the gradient! 1. The stability of the lock system is critical for many experiments. NOE measurements. This is achieved by delivering a suitable current to the z0 shim coil. one can determine how fast the lock circuit reacts (the damping of the lock). the carrier frequency. all other resonance frequencies are also kept constant. Bottom: Signal due to positive frequency. Usually the lock system has to be activated when the sample has been placed in the magnet.
the frequency with which the B1 ﬁeld rotates. Sineand cosinemodulated components of the signal result from a different trick: The signal that comes from the receiver coil (which of course is HF (MHz)) is split into two.modulated and the other the sinemodulated component of the signal.y plane and in the receiver system by subtracting the transmitter frequency from the signal. Sign discrimination from quad. Adding sine.29
. such a separation could be achieved by using two receiver coils which differ by 90° in phase (a quadrature detector):
b) quadrature detector
ν
0
+ν
ν
FIGURE 8. that both the transmitter and the receiver system effectively work in the rotating frame. The rotating frame was introduced in the transmitter system by having the B1 ﬁeld rotating in the x. What we actually measure are therefore not MHzfrequencies but frequencyoffsets (differences to) the carrier frequency. These can be handled in the following electronics much easier. probeheads contain only one coil. Thereby. We do see now. In addition. The signal coming from
Second Chapter: Practical Aspects Pg. we have introduced quadrature detection: Transmitting and receiving is done on the same coil. the phase of the transmitter frequency that is added differs by 90 degrees for the two parts.LECTURE COURSE: NMR SPECTROSCOPY
In principle.and cosine modulated components of the signal cancels the “wrong” frequency component:
FT
sin ωt
cos ωt
FT
Σ
FIGURE 9. Fourier transform of a sin (top) and cosine wave (bottom). detection
0
+ν
One could would then detect the cosine.
However. However. the radiofrequencies (MHz range) are transformed into audiofrequencies (kHz range). Both parts are mixed with the transmitter (carrier) frequency [for Bruker machines: SFO1].
LECTURE COURSE: NMR SPECTROSCOPY
the coil is blanked for a short time after the pulses (the socalled prescan delay) and then used for detection. The transmitter/receiver system contains mainly analog parts.
Splitter 90° paseshifter
HF (MHz)
cos ωt
sin ωt
Mixer
SFO1 (300.13 MHz)
+
Amplifier AnalogDigital Converter ADC
Audio ( 0KHz)
FT
FIGURE 10. Scheme of the RF path for a 300 MHz NMR spectrometer (proton detection).
When cosine and sinemodulated signals are slightly differently ampliﬁed socalled quadrature images remain. They are usually observed for intense signals when few scans are recorded and can be easily recognized because they are mirrored about the
Second Chapter: Practical Aspects Pg.30
LECTURE COURSE: NMR SPECTROSCOPY
zerofrequency (the middle of the spectrum). Therefore, modern instruments utilize “oversampling”. A much larger spectral width is sampled, such that the quadrature images do not fall into the observed spectrum any longer. Prior to processing the noninteresting part of the spectrum is removed.
2. BASIC DATA ACQUISITION PARAMETER
The following paragraph summarizes the parameters that govern the acquisition of 1D spectra and hence have to be properly adjusted before the measurement. The 1D spectrum is characterized by the frequency in the center of the spectrum and by its width. Remember that the B1 ﬁeld rotates about the axis of the static ﬁeld with a certain frequency:
Bo
ω ω0 y x B1
ω Bo ω0
ω1
FIGURE 11.
Exactly this frequency is mixed with the signal that comes from the receiver coil and therefore is effectively subtracted from the signal frequency. Hence, the signal is measured in the rotating frame and the frequencies will be audio frequencies (0 kHz). If the precession frequency of a particular spin is exactly the same as the frequency with which the B1 ﬁeld rotates it will have zero frequency and appear in the center of the spectrum. The frequency of the rotating B1 ﬁeld is called the (transmitter) carrier frequency:
spectral width (sw)
 sw/2 0 transmitter frequency (sfo1) + sw/2
FIGURE 12. Basic parameter for specifying the acquired spectral region
sfo1: spectrometer frequency offset of the ﬁrst (observe) channel this parameter determines the carrier offset. Frequencies larger than sfo1 will be on the left side of the spectrum, smaller frequencies on the right side. sfo1 is the absolute frequency (in MHz). If you want to make a small change of the carrier position, you can change the offset o1
Second Chapter: Practical Aspects Pg.31
LECTURE COURSE: NMR SPECTROSCOPY
sfo1 = bf1 + o1 bf1 is the socalled basicspectrometer frequency, e.g. 500.13 MHz and o1 is the offset, a frequency in the audio (0> KHz) range. sfo2: spectrometer frequency offset of the second (decoupler) channel like sfo1, required for example for acquisition of proton decoupled carbon spectra. The proton frequency is then deﬁned on the second channel and should correspond to the center of the proton spectrum. Now, the next question is, how to adjust the spectral width (the width of the spectrum in Hz). This done by proper choice of the dwell time. The signal is sampled stroboscopically and neighboring data points are separated by the dwell time. The Nyquist theorem says that at least one point per period must be sampled in order to characterize the frequency correctly. Since the highest frequency is sw (in Hz), the dwell time must be dw = 1/sw
(td =16)
DW
AQ
FIGURE 13. Left: Sampled data points. Right: Black: Sampled data points. Red: Missing sampling point for a frequency of twice the nyquist value..
Signals that have a higher frequency have made more than a 360 degree rotation between the sampling points. On older instruments (AMX spectrometers) they are attenuated by the audioﬁlters. They are recognized from the fact that they cannot be phased properly, usually. On the modern instruments (those with oversampling, DRX series), signals outside the spectral width completely disappear. Therefore, it is always recommended to record the spectrum over a very large spectral width initially to make sure that no signals at unusual places (e.g. OH protons at 20 ppm or signals below 0 ppm) are lost.
Second Chapter: Practical Aspects
Pg.32
If aq is set to a shorter value. aq: acquisition time determines the total time during which a single FID is sampled aq = td * dw for 1D spectra. dw: dwell time is the time spacing between sampling of two consecutive data points. dw = 1/sw or dw = 1/2sw The dwell time is determined by the inverse of the spectral width (or the inverse of twice the spectral width. td: time domain data points The number of points that are sampled is called "time domain" data points (td). if aq is >> T2. depending on the quadrature detection mode). and therefore the experimental time is unnecessarily prolonged. the minimum number of ns is determined by the phasecycle of an experiment.LECTURE COURSE: NMR SPECTROSCOPY
sw: spectral width gives the width of the acquired spectrum (on Bruker instruments. ttot = ns (RD+aq) in which ns is the number of scans and RD the relaxation delay (vide infra). The longer the FID is sampled the better will be the resolution in the spectrum provided there is still signal in the FID. RD: relaxation delay time between end of sampling of the FID and start of the next experiment. the relaxation delay is characterized by the parameter d1.33
. The effective time for relaxation is given by the repetition rate
Second Chapter: Practical Aspects
Pg. On Bruker instruments. sw is in units of ppm. the resolution is lower. only noise will be sampled at the end of the FID. The signal to noise ratio of a measurement N increases as which means that in order to improve the S/N by a factor of two the measurement time must be increased by a factor of 4. swh in units of Hz). aq is set such that the signals have completely decayed towards the end of the FID (which corresponds to the transverse relaxation time T2). ns: number of scans S ∝ ns the number of FIDs which are coadded. Sometimes. The complete measuring time for a simple 1D spectrum is approx.
p1... We have seen before that if the frequency ω with ωo=SFO1 which the spin precesses about the zaxis is identical to ωo it does not feel B0 in the rotatω1=(4*(PW90) ) 1 ing frame.p2..It will then precess about the axis of the effective ﬁeld which in that case corresponds to the axis of the B1 ﬁeld. 1. If the receiver setting is too low. If the receiver gain setting is too high. The following conventions are made in pulseprograms: • • • • p190˚ transmitter pulse length p2180˚ transmitter pulse length p390˚ decoupler pulse length p4180˚ decoupler pulse length
Pg.32k).256.length of pulses [in µs]
ω Bo
length of pulses in µs. it is in the kHz range (or smaller)... is much smaller.g. ω1. The strength of the B1 ﬁeld is correlated to the time required for a 360 degree rotation PW360 via ω1= γB1 / 2π = 1/(PW360) Usually either 90 or 180 degree rotations are required in NMR.34
Second Chapter: Practical Aspects
. However. This is the duration that the B1 ﬁeld is switched on. For automatic adjustment of the receiver gain on spectrometers use rga. Settings are possible in powers of 2 (e.
D1
rg: receiver gain This is the ampliﬁcation of the signal. the ﬁrst points of the FID are clipped. which will lead to severe baseline distortions.LECTURE COURSE: NMR SPECTROSCOPY
tr = aq + d1
AQ tr
FIGURE 14.128. The receiver gain has to be set such that the maximum intensity of the FID ﬁlls the ADC (analogdigital converter). the dynamic range of the instrument is not exploited fully and very small signals may be lost.. Definition of the recycle delay.. the precession frequency about the B1 axis. The B1 ﬁeld rotates
about the zaxis with the frequency ωo..
.).tl6 (transmitter level 1.
Second Chapter: Practical Aspects
Pg.tl1.dl1.hl3. On the newer DRX instruments the power levels are speciﬁed as pl1:f1 (power level 1 on channel 1).dl6 (decoupler level 1.. In the default mode.. 15N.LECTURE COURSE: NMR SPECTROSCOPY
The ﬂip angle of the pulse Φ is determined not only by the length of the pulse τp. but also by the power level.hl6 (hl= 1H level). For pulses on the heteronuclei (13C.2.. 31P ..2.) for the low power mode.. heteronuclear pulsing is done in the highpower mode and cannot be regulated (always full power)..35
.hl2. power setting parameters are tl0. the power level is given by the parameters hl1.) or dl0... at which the B1 ﬁeld is applied: Φ = τp γB1 For proton pulses.
36
. as well as for the 360˚ pulse.LECTURE COURSE: NMR SPECTROSCOPY
1. The length of the pulses has therefore to be determined for a given power setting. phasing of the spectrum. 20 ppm) acquisition of the final spectrum with sufficient S/N and optimized spectral width
•
• • • • • • •
•
1. For calibration of proton pulses. When the length of the pulse is set corresponding to a 180˚ pulse. higher power setting must be applied (typically for 1H: 50 W. a zerocrossing of the signal is observed. approx. The length of the 90˚ pulse can be calculated to be half of the length of
Third Chapter: Practical Aspects II
Pg. for 15N:300W). a spectrum is acquired with setting the length of the pulse corresponding to a small ﬂip angle. and then increasing the pulselength in small steps.1 Calibration of pulse lengths: The ﬂipangle θ of pulses is proportional to the duration (τp) and strength of the applied B1 ﬁeld:
θ = γB1τp
It is also obvious that for nuclei with lower γ. ACQUISITION OF 1D SPECTRA
Acquisition of simple 1D spectra comprises the following steps • • • changing the probehead Setting the correct temperature for measurement Correct positioning of the tube in the spinner and insertion of the sample into the magnet recalling a shimset with approximately correct shims for the solvent and probehead activation of the field/frequency lock system adjustment of the homogeneity of the magnetic field (shimming) recalling a parameter set for the desired experiment (tuning of the probehead) (calibration of the pulse lengths) determination of the correct receiver gain setting performing a preliminary scan with large spectral width (1H only.
Calibration is performed in the socalled inversedetection mode with the following sequence detecting the proton signal (which is split by the 13C coupling into an antiphase doublet):
1H
1/2J(C.LECTURE COURSE: NMR SPECTROSCOPY
the 180˚ pulse:
FIGURE 1. and no signal is observed:
15us
10 us (90 deg.2.
Third Chapter: Practical Aspects II
Pg. Calibration of pulse length. all magnetization is converted into multiplequantum coherences.H) 13C
FIGURE 2.
13C
pulses are most easily determined on a 13Clabeled sample such as an acetate sample (labeled at the Me carbon). Pulse sequence to determine the 90° carbon pulse with proton detection
When the carbon pulse is exactly set to 90˚. The length of the proton pulse is incremented by a fixed amount for each new spectrum.H)
FIGURE 3.37
. Resulting spectra from the sequence of Fig.)
5us 1us
J(C.
The reason for this is. Care has be taken when solvents with high salt contents are used: Even in the case of perfect matching and tuning the pulselengths can be considerably longer (about 1. The energy is interchangingly stored in form of an electric or magnetic ﬁeld in the capacitor or in the coil.38
.LECTURE COURSE: NMR SPECTROSCOPY
1. although this effect largely depends on the nucleus and is most pronounced for proton frequencies. Tuning and matching can be performed in different ways: (A) the reﬂected power
Third Chapter: Practical Aspects II Pg. L). that if the sample is introduced into the inner of the coil the dielectric constant in that part and hence the impedance of the coil changes.
When properly tuned and matched.5 times for protons when using 150mM NaCl). Basic principle of the probehead circuit. An oscillating electromagnetic ﬁeld is also part of a radio and hence the resonance circuit inside the probe has much in common with the one found in a radio:
L C E B
FIGURE 4. A large inﬂuence to the tuning and matching is caused by the dielectric constant of the sample. respectively. In order to get best sensitivity and the shortest pulselengths the probe has to be • •
13C
tuned to the transmitter frequency the impedance should be matched to 50 Ω.
A resonance circuit has two basic components: a capacitor (C) and a coil (or inductance.2 Tuning the probehead: The purpose of the radiofrequency circuit inside the probe is to deliver a rotating B1 ﬁeld and to detect the signal. The frequency of oscillation is ω = 1 ⁄ ( LC ) in which L is the inductance of the coil and C the capacity. the probe has optimum sensitivity and the pulse lengths for example do not have to be measured on the sample but predetermined values can be used instead.
and the inner one has higher sensitivity.39
510
.
Modern probes usually have two coils. and consequently. Thereby.
Third Chapter: Practical Aspects II Pg. 1. Wobbling curve of a detuned probe (thick line). an inner coil and a outer coil. the minimum is moved along the horizontal axis and by matching the minimum becomes deeper. the thermal noise level is largely reduced (the amount of signal is even a little less than for a conventional probe) and the signal/noise in the absence of salt increased by almost a factor of four.3 Adjusting the bandwidth of the recorded spectrum: The bandwidth of the spectrum is determined by the dwell time (dw). The dual probe has higher sensitivity for 13C. Arrangement of coils for inverse and direct carbon detection probeheads
Recently. (B) Nowadays wobbling is used during which the frequency is swept continuously through resonance covering a bandwidth of 420 MHz:
matching
tuning
490
SFO1
FIGURE 5. the time spacing between recording of two consecutive data points (vide supra). which is optimized for 1H detection has 1H frequency on the inner and 13C frequency on the outer coil. By tuning.LECTURE COURSE: NMR SPECTROSCOPY
of the probe during pulsing can be minimized. socalled cryoprobes have been introduced. For these probes the RFcoil and the preampliﬁer is at Hetemperature. the inner coil is used for 13C:
≈ ≈
Inverse Dual
1H 13 C 13C 1H
FIGURE 6. An inverse probe. The optimum setting is shown as a dotted line.
when the offset is varied slightly. The Fourier theorem states that every periodic function may be decomposed into a series of sine. signals may easily become lost. Usually 10% are added to the edges because the audio ﬁlters tend to decrease signal intensity of signals close to the edge. Although analog or digital ﬁlter decrease the intensity of folded peaks. The best way to prevent folding or loosing of signals is to record a preliminary experiment with a very large bandwidth (e.40
.and cosine functions of different frequencies:
∞ ∞ n=1
f ( x ) = a0 ⁄ 2 +
∑ an cos nx + ∑ bn sin nx
n=1
The Fourier decomposition therefore tells us which frequencies are present to
Third Chapter: Practical Aspects II Pg.
FIGURE 7. Effect of folding (aliasing) of signals.4 Data processing: Once the analog signal that comes from the coil has passed the ampliﬁers.LECTURE COURSE: NMR SPECTROSCOPY
If the sampling rate is too low. time [f(t)] are then converted into a spectrum that represents signal intensity vs.g. if the spectral width is set too small. they may well be in the spectrum. Folded peaks can be recognized. because usually they are not inphase after adjustment of zero and ﬁrst order phase correction. frequency [f(ω)] by use of the Fourier Transformation. they change their position in the spectrum. care has to be taken on the newer instruments that use oversampling and digital filters.
However. it is converted into a number by the analogtodigital converter (ADC). The resulting numbers which represent signal intensity vs. the signal will still be sampled but will have a wrong frequency after Fourier transformation. Alternatively. 30 ppm for 1H) and then adjusting offset and spectral width to cover all signals. In this case the frequencies outside the sampled region will be almost completely removed by the digital filters! 1. On the other hand.
which transforms a Npoint time series consisting of values dk into a Npoint spectrum f:
N–1
fn = 1 ⁄ ( N )
∑ dk e
k=0
– 2πikn ⁄ N
The discrete FT is implemented in form of the very fast CooleyTukey algorithm. Analogously.into the timedomain:
∞
F ( t ) = 1 ⁄ ( 2π )
∫ f ( ω )e
–∞
– iωt
dω
Note that this transformation is the continuous FT (used for continuous functions). Hence.LECTURE COURSE: NMR SPECTROSCOPY
which amount (with which intensity). the integral is split up again into the cosine. The consequence of using this algorithm is.
Third Chapter: Practical Aspects II Pg. Usually. the discrete FT has to be utilized. the FT can be used to transform from the frequency.and sine terms corresponding to the cosine and sine transforms. that the number of points the spectrum has must be a power of 2 (2n). Using the integral form of the equation above
∞
f ( ω ) = 1 ⁄ ( 2π ) a ( t ) = 1 ⁄ ( 2π ) b ( t ) = 1 ⁄ ( 2π )
∫
–∞ ∞
a ( t ) cos ( ωt ) + b ( t ) sin ( ωt ) dt
∫ f ( x ) cos ( tx ) dx
–∞ ∞
∫ f ( x ) sin ( tx ) dx
–∞ ∞ – iωt
and utilizing the Euler relation the equation can be rewritten as: f ( ω ) = 1 ⁄ 2π
∫ F ( t )e
–∞
dt
This is the recipe to transform the signal function F(t) which for a single resonance is F(t) = e
– iωt – t ⁄ T2
•e
into the spectrum F(ω). so that
∞
∫ f ( t )e
–∞ ∞
iωt
dt = Re [ f ( ω ) ] + Im [ f ( ω ) ]
∞
=
∫ f ( t ) cos ωt dt + i ∫ f ( t ) sin ωt dt
–∞ –∞
Note that in FTNMR the signal is sampled as discrete points.41
.
A gaussian function yields a gaussian function after FT.
The FT of a cosinewave gives two deltafunctions at the appropriate frequency (A). The FT of a step function gives a sinc function sin c ( x ) = sin ( x ) ⁄ x The sinc function has characteristic wiggles outside the central frequency band (D). In fact the FT of (C) can be thought of a convolution of (A) and (B).42
. Results of different functions after Fourier transformation in frequency space. The FT of a properly shimmed sample containing a single frequency gives two signals at the appropriate frequency with lorentzian lineshape (C).LECTURE COURSE: NMR SPECTROSCOPY
The next ﬁgure will summarize the FT of some important functions (the socalled Fourier pairs):
A
B
C
FT
D
E
F
FIGURE 8. The FT of a decaying exponential gives a lorentzian function with a characteristic shape (B). Signals that have not decayed to zero are common in 2D
Third Chapter: Practical Aspects II Pg. A FID that has not decayed properly can be thought of as a convolution of a step function with an exponential which is again convoluted with a cosine function. The FT gives a signal at the appropriate frequency but containing the wiggles arising from the sinc function (E).
the line shape will be purely dispersive:
Third Chapter: Practical Aspects II
Pg. respectively. real and imaginary parts of the signal are stored in separate memory locations. Signal in pure absorption or dispersion mode
Purely absorptive signals have a much narrower base. the real part that is used for displaying the spectrum does not necessarily need to be purely absorptive:
1/ π T2
dispersion mode
absorption mode
FIGURE 9. a phasecorrection φ is needed to yield purely absorptive signals. If the signal starts as a pure sinemodulated oscillation. If the oscillation of the signal of interest is purely cosinemodulated. the signal will have pure absorptive line shape.LECTURE COURSE: NMR SPECTROSCOPY
NMR were the number of data points sampled is restricted. However. Therefore.5 Phase Correction We have seen that the complex Fourier transform can be decomposed into a cosine transform and a sine transform that yield the real and imaginary parts of the signals. The phase of the signals after Fourier Transformation depends on the ﬁrst point of the FID. respectively. so that differentiation of peaks very close to each other is easier if they have been phased to yield purely absorptive line shape. 1. In practice. It will do the following manipulations: Re ( ∆Φ ) = – Im ( ∆Φ = 0 ) sin φ + Re ( ∆Φ = 0 ) cos Φ Im ( ∆Φ ) = Re ( ∆Φ = 0 ) sin φ + Im ( ∆Φ = 0 ) cos Φ with Re(∆φ=0) and Re(∆φ) being the real part of each data point before and after the phase correction.43
. In these cases suitable window functions are used to force the signal to become zero towards the end of the FID.
A) Ideal FID. In a theoretical 1D experiment.
Third Chapter: Practical Aspects II
Pg. Top: The zero order phase correction changes the signal phase for all signals by the same amount. in a real experiment there is a protection delay (called de on Bruker instruments). to wait for the pulse ringdown (pulsing and detection of the signal is done on the same coil. acquisition of the FID would start immediately after application of the pulse. The linear dependence of the phase of the signals has a more complicated reason.
What is the origin of the varying phase in the spectra? The zeroorder arises because the absolute phase of the signals at the detector depends on the cable lengths etc. The relative phase of the magnetization with respect to the receiver coil determines the phase of the signal after Fourier transformation
There are two different types of phasecorrection. so that there must be a protection delay after the pulse):
A
B
DE
FIGURE 12.LECTURE COURSE: NMR SPECTROSCOPY
t=0
t=0
FIGURE 10.44
. The zeroorder phase correction applies the phase change to all signals in the same way. B) Introduction of the prescan delay to enable pulse power ringdown. However. Lower: For the first order phase correction the applied correction depends on the frequency difference to the reference signal (marked by an arrow). whose amount increases linearly with the distance to the reference signal (marked with an arrow in the ﬁgure below):
PC "0 th order"
PC "1 st order"
FIGURE 11. The ﬁrst order phase correction applies a phase change.
When spectra cannot be phased a magnitude calculation or the power spectrum (PS=M2) is used:
Third Chapter: Practical Aspects II Pg. use the zeroorder phase correction to phase it to absorption and then use the ﬁrst order phasecorrection to phase the signal at the other edge of the spectrum. (A) normal FT with phase correction. (C) Magnitude calculation. e. The precession frequency depends on the offset of the signal frequency. deﬁne a signal at one end of the spectrum as the reference phase.45
. The ﬁrstorder phase correction reverses the phase change due to shift evolution during de and is therefore linearly dependent on the frequency of the signal:
A B
≈ ≈
B
FIGURE 13.
To adjust the phase.g. go into the phase correction mode. All other but folded signals will then be in phase. Precession of spins with different frequencies during the prescan delay.(B) Power spectrum.LECTURE COURSE: NMR SPECTROSCOPY
During the protection delay de.
FIGURE 14. the distance of the signal from the middle of the spectrum. the spins start to precess.
the number of points which are used to digitize the signal. 1.6 Zeroﬁlling and the resolution of the spectrum The resolution that can be obtained from a spectrum is determined by two factors: • the natural line width that is related to the transverse relaxation time (homogenous broadening) and which is due to efficient dipolar coupling of the spins. and the resolution is very low:
•
FIGURE 15. Fast transverse relaxation may also be caused by field inhomogeneity (inhomogenous broadening). If the number of points after FT (on Bruker instruments the parameter SI) is smaller than the number of acquired data points (on Bruker instruments the parameter TD). Right: Spectrum without zerofilling (lower trace) and after zero filling with 5*TD (upper trace). magnitude and power calculation lead to a severe loss in resolution. the signal can not be digitized properly.
The theoretical resolution obtainable from the FID can be calculated as Res = SW (Hz) / TD neglecting relaxation effects. Left:Effect of increasing number of data points after FT. Both. Provided that the FID has decayed to zero towards its end it is useful to
Third Chapter: Practical Aspects II
Pg.LECTURE COURSE: NMR SPECTROSCOPY
M =
Re + Im
2
2
The magnitude calculation yields much broader peaks at the base.46
.
Application of the window function amounts to multiplying the FID data point dk by dk’=dk*ak in which k runs from 0 to TD (the total number of data points). but good resolution is required. and data could be multiplied with an increasing exponential (em with LB< 0).LECTURE COURSE: NMR SPECTROSCOPY
append zeros to the FID. Most manipulations that improve resolution lead to a loss in S/N. There are number of socalled window functions that are aimed at either improving resolution or S/N and their effect on a spectrum is shown in the ﬁgure below:
Third Chapter: Practical Aspects II Pg. Any data manipulation. Top: Resulting FT of a FID that did not decay to zero in comparison to a fast decaying FID (bottom)
1. S/N is comparably bad. Data processing of 13C spectra therefore usually is performed with exponential multiplication with a decaying exponential (em with LB>=1). Therefore. S/N is rarely a problem. whereas resolution is rarely a problem. When the FID has not decayed to zero. the resolution is determined by the amount of signal which remains towards the end of the FID. e.47
. the S/N is determined by the amount of signal at the beginning of the FID. Adding zeros once (that means making si = 2*td) gives a signiﬁcant improvement in resolution. fast relaxation to a quickly decaying FID. adding zeros introduces a step function that causes sinctype artifacts (wiggles). A long transverse relaxation time leads to a slowly decaying.7 Resolution enhancement and S/N improvement The resolution is determined by the transverse relaxation time T2 of the spins. Conversely.g. In 13C nmr.
FIGURE 16. In 1H nmr. multiplying the FID with a increasing exponential (et/x) will improve the resolution. adding more zeros gives only cosmetic effects but does not add more information.
ak = e
– πlbk∆t
1.1 Exponential multiplication: The line broadening constant lb can be positive (sensitivity enhancement) or negative (resolution enhancement). whereas gb determines the center of the maximum of the gaussian curve. When gb is set 0. A lorentzian line of width W will have a width of W + LB Hz after apodization.(F)90 degree shifted sinebell.7. (D) sinebell. but LB=1.(E) 45 degree shifted sinebell. the maximum of the
Third Chapter: Practical Aspects II Pg. (B)Multiplication with exponential. ak = e
πlbk∆t
•e
– gb ( k∆t )
2
The factor lb determines the broadening.48
.LECTURE COURSE: NMR SPECTROSCOPY
FIGURE 17. frequently used window functions are 1. It is mainly used for processing 13C data. (A) raw spectrum after FT.33.LB=5.7.2 LorentztoGauss transformation: This a combination of exponential and gaussian multiplication.(C) as (B). for which sensitivity enhancement is desired but where resolution is rarely a problem (use lb = 12 Hz).
Important.
7. This is usually used for FIDs that have maximum signal in the ﬁrst data points (NOESY. A pure sinebell corresponds to the ﬁrst halflobe of a sinewave and multiplies the FID such that it brought to zero towards the end. By this multiplication.3 SineBell apodization: Sinebell apodization is frequently used in 2D NMR processing.LECTURE COURSE: NMR SPECTROSCOPY
function occurs after 1/3 of the acquisition time. Left: Lorentzian lineshape. A pure sinebell is used for such experiment in which the FID does not have maximum signal in the ﬁrst points (such as COSY) or for improving magnitude data (HMBC):
FIGURE 19. However. which has a narrower base:
FIGURE 18. such a function also sets the ﬁrst data points to zero and hence leads to severe loss of S/N and introduces negative wiggles at the bases of the signals in addition to a strong improvement in resolution.Right: FID of NOESY with cosinebell
Third Chapter: Practical Aspects II
Pg. Left: FID of COSY with shifted sinebell.49
. the Lorentzian lineshape is converted into a gaussian lineshape. Right: Gaussian lineshape. HSQC).
1. Shifting the sinebell by 90 degrees (a cosinebell) means that the function used starts of the maximum of the ﬁrst lobe and extends to zero towards the end of the FID.
LECTURE COURSE: NMR SPECTROSCOPY
The inﬂuence of the parameters on the window functions is shown below:
FIGURE 20. that the receiver gain has been set too high.1. e. in spectra with baseline roll small signals may not be recognized. 1. If the signal intensity is larger. spectra with nonﬂat baselines give wrong integrals. that the signal is not sampled until it has completely decayed. Then the signal can be described by a superposition of an oscillation. Firstly. must be kept to a reasonable low value. The contribution from the square function will introduce wiggles at the base of the signals which are originating from the sinc function. when the data matrices. Furthermore. that means. This is the case. A: exponential multiplication with LB=1. e. In general. baseline distortions are caused by distortions of the ﬁrst points of the FID.g.g. π/2. A frequent source is receiver overload. 0.
An often encountered problem is truncation of the signal.3. which means. that means that the FID must ﬁt into the acquisition window completely. B) LorentzGaussian transformation with LB=5 and GB =0. There are many reasons for bad baselines. π/2. π/4. The ADC (analogtodigital converter) can only digitize a certain maximal signal intensity. an decaying exponential or a cosine bell function. in 2D NMR. π/8.5 Hz. π/8. the number of data points. Secondly. or when one very slowly decaying line is present in the spectrum.50
. e.g. Different window functions.8 Baseline Correction Baselines that are not ﬂat are a major problem in FTNMR. Thirdly. peakpicking routines need a threshold for minimal signal intensity and such peakpicking for tiny signals is not possible when the baseline is not ﬂat. D) Sinebell squared shifted by 0. On the Bruker instruments. C) Sinebell shifted by 0. Such a problem can be circumvented if the FID is multiplied with a function that decays to zero at the end of the FID.3. the ﬁrst points of the FID are clipped:
Third Chapter: Practical Aspects II
Pg. 0. those spectra are difﬁcult to phase. π/4.5Hz. an exponential and a square function.
A wrong ﬁrst data point leads to an offset. Predicting more points severely changes the lineshape of the signals and is not recommended. Frequently. spectra with (a few) wrong ﬁrst data points can still be used. Top: Spectrum after FT of a clipped FID. a wrong third one to a full wave.
Such a baseline cannot be corrected. on Bruker machines either automatically (with the command abs) or manually. Especially in the case of the
Third Chapter: Practical Aspects II Pg. a wrong second one to a halfwave. like baselinedrift (nonhorizontal but ﬂat baseline) or baselinecurvature can be corrected. 1. In principle.LECTURE COURSE: NMR SPECTROSCOPY
FIGURE 21. The reason behind this is that one can assume that new data points can be represented as a ﬁxed linear combination of immediately preceding values. Other baseline problems.9 Linear prediction: For exponential decaying signals that are sampled at constant spacing (as is usually the case for NMR) new data points can be predicted. if linear backprediction of the ﬁrst data points is used. and the measurement must be repeated with a lower receiver gain setting.51
. LP provides a means of ﬁtting a time series to a number of decaying sinusoids. LP gives very good results for COSY and HSQC data. Oversampling has shown to reduce baseline problems in the directly sampled frequency dimensions dramatically. Provided that S/N is good the number of points can be doubled with LP (or the measuring time can be halved to achieve the same resolution). etc. lower trace: spectrum after FT of an FID with correctly adjusted receiver gain. one can recognize which data point is distorted from the shape of the baseline.
Remember that in order to double the resolution in the indirect dimension the number of experiments in a 2D spectrum and hence the acquisition time has to be doubled.(C) LP to 128 points.52
. mirrorimage linear prediction may be applied which is capable to predict N points to 4*N points.
Third Chapter: Practical Aspects II
Pg.LECTURE COURSE: NMR SPECTROSCOPY
COSY. Linear prediction in these cases not only increases the resolution but also increases the amount of signal! With modern computing power it is performed rather quickly and the results can be astonishing good.
FIGURE 22. Linear prediction may achieve the same goal in a few seconds. In the case of constanttime experiments.(D) mirror image LP to 240 points. antiphase signals cancel when the resolution is low. ctHSQC spectra (A) 240 points.(B) 64 points.
LECTURE COURSE: NMR SPECTROSCOPY
1.spin couplings. Many different mechanisms are known that may inﬂuence the exact strength of ﬁeld at the nucleus: σ = σdia(local) + σpara(local) +σm + σrc + σef + σsolv Herein is • • • • • • σdia the diamagnetic contribution σpara the paramagnetic contribution σm the neighbor anisotropy effect σrc the ringcurrent contribution σef the electric ﬁeld effect σsolv the solvent effect
1. What is really important is the effective ﬁeld strength at the nucleus site. the resonance frequency depends on the strength of the applied static ﬁeld and on the gyromagnetic ratio of the nucleus:
ω = γB
However. this splitting of lines is caused by the chemical shift due to the Zeeman interaction and to scalar spin.1 The diamagnetic effect: The nucleus is surrounded by an electron cloud.53
. a current is created that itself builds up a magnetic ﬁeld. In liquid (isotropic) phase. The direction of the induced ﬁeld is opposed to the static ﬁeld and acts to decrease the strength of the latter:
Fourth Chapter: Chemical shifts and scalar couplings
Pg. The static magnetic ﬁeld Bo causes the electrons to precess about the axis of the magnetic ﬁeld. As we have seen before. the ﬁeld strength to be considered is not exactly the strength of the applied magnetic ﬁeld in vacuo but is locally modiﬁed by the electronic environment of the nucleus. Thereby. THE CHEMICAL SHIFT:
Soon after the discovery of the phenomenon of nuclear magnetic resonance scientists realized that more than a single line was observed for protons in a organic molecule such as ethanol.
Quantummechanically speaking.LECTURE COURSE: NMR SPECTROSCOPY
Bo
FIGURE 1. the paramagnetic term may be calculated by ﬁnding the way by which the electron wave functions are modiﬁed by the magnetic ﬁeld. e the charge and me the mass of the electron.54
. 1. The diamagnetic shielding describes the behavior of spherically distributed electrons such as electrons from the sorbital of protons.2 The paramagnetic term: The paramagnetic term serves to correct for the disturbance of the spherical rotation of electrons. Such a modiﬁcation of the wave function takes place because properties
Fourth Chapter: Chemical shifts and scalar couplings
Pg. It is therefore the dominant term for proton shifts but less important for the heavier nuclei. It can be calculated as
∞
σ iso = µ 0 e ⁄ 3m e • ∫ rρ ( r ) dr
0
2
in which ρ(r) is the electron density. One important statement of this socalled BiotSavart Law is that the electron density at larger distances from the nucleus is of importance for the counteracting ﬁeld.
This effect is called the Lamb Shift. r the distance of the electron from the nucleus. This may be caused by the formation of bonds.
For 13C it is given as σi
para
= ( µ 0 µ B ) ⁄ 2π∆E 〈 r 〉 Q i +
2
–3
i≠j
∑ Qj
In this formula.55
. highest occupied molecular orbital) functions through application of the static ﬁeld. It is the dominant term for all nuclei other than protons. Substituents inﬂuence the chemical shift because they change the electron denFourth Chapter: Chemical shifts and scalar couplings Pg. 15 ppm for 1H). Mixing of excited state wavefunction into groundstate wavefunctions through the Bo field. ∆E is the energy difference between HOMO and LUMO . Since the paramagnetic shift is inversely proportional to the energy gap. r the radius of the 2p orbital and the Q describe electron densities and bond orders. the HOMO. The shift range due to the paramagnetic contribution is larger than for the diamagnetic shift. This is obvious from the huge shift ranges (e. several thousand ppm for 57Fe compared to approx.
The calculation of the diamagnetic shift is rather involved and requires the exact knowledge of the excited wave functions.g. lowest unoccupied molecular orbital) are mixed into ground state wave (esp. Very lowlying groundstate wavefunctions are usually paired with rather high lying excited state wave functions such that the resulting energy separation is comparably large. The electron distribution in such an excited state wavefunction is very different from that encountered in the ground state giving rise to a large change in the shielding:
LUMO Bo HOMO
FIGURE 2. Reactivities of compounds are often related to HOMO/LUMO energy separations and chemical shifts have therefore been successfully used to screen for reactive compounds. the shift is large when the gap is small.LECTURE COURSE: NMR SPECTROSCOPY
of exited states (contributions from the LUMO. The paramagnetic term requires nonspherical electron distribution (a nonvanishing angular momentum) and can hence only be applied to nuclei that have nons orbital electrons.
1 ca 12 ppm
O
H
e.r. The magnitude of the shielding therefore depends on the relative orientation of the nucleus with respect to the static ﬁeld. mat.= 2.3 Chemical shift anisotropy: The distribution of the electrons about the nucleus is usually nonspherically. to external field
The chemical shielding is described by the chemical shift tensor. For sp3 carbons the nuclei are tetrahedrally coordinated and the electron distribution is almost invariant under rotation. Due to the shielding the direction of the effective ﬁeld Beff may deviate from the direction of the static
Fourth Chapter: Chemical shifts and scalar couplings Pg.5
e.5
H
e.56
.) The zz axis is usually taken as the axis with the largest shielding.1 ca 4 ppm
FIGURE 3. This is fairly evident from a comparison of a oxygen and a carbon bound proton:
C
e.= 3.LECTURE COURSE: NMR SPECTROSCOPY
sity at the proton nucleus via inductive effects. Dependence of chemical shielding upon orientation of carbonyl bond toi the external field
1.= 2. which transforms the static ﬁeld into the effective ﬁeld (see add. Shielding of carbonyl carbon in dependence of orientation of zzaxis w. For sp2 carbons such as aromatic or carbonyl nuclei the shielding highly depends on the orientation of the bond relative to the static ﬁeld:
σzz σzz
C
O
C
O
FIGURE 4.= 2.
LECTURE COURSE: NMR SPECTROSCOPY
ﬁeld Bo. An example is a triple bond. In liquids this effect contributes to relaxation. The π electrons of the triple bond form an electron cloud that extends around the bond axis in form of a tube. Chemical shift anisotropy values can be measured from solidstate NMR. A similar counteracting ﬁeld is formed in the πcloud of aromatic systems.4 Magnetic anisotropy of neighboring bonds and ring current shifts: Some types of neighboring bonds create an additional magnetic ﬁeld which is anisotropic in space. However. They usually increase the dispersion of proton spectra. in order to give substantial effects the inﬂuenced protons
Fourth Chapter: Chemical shifts and scalar couplings Pg. The magnetic ﬁeld forces the electrons to rotate about the bond axis creating a magnetic ﬁeld whose direction is along the bond axis and which again counteracts the static ﬁeld. xx and yy orthogonal to zz. The zz axis points along the value
of the largest shielding. Right: Direction of the static and the effective field.
σzz
Bkorr Bo Beff
σxx σ yy
FIGURE 5. the line is signiﬁcantly broadened. as is the case in the solid state. Such anisotropies can dramatically change the appearance of proton spectra. Since the molecule rotates quickly in solution the observed chemical shift is a average over the shifts coresponding to the different orientations (isotropic chemical shift). when rotation is hindered.57
. However. and is the major relaxation mechanism for nonprotonated carbons.
The value of the chemical shift for the isotropic case can be taken as 1/3 of the trace σ iso = 1 ⁄ 3 ( σ xx + σ yy + σ zz )
1. Left: Coordinate system used for the chemical shift tensor.
58
.
Bo Bo
C
C
e
e
FIGURE 6. depending on the exact postion of the proton.
Fourth Chapter: Chemical shifts and scalar couplings Pg. Aliphatic Protons that are ﬁxed in space above the plane of an aromatic ring can be shifted to values below 0 ppm. Therefore. The direction of the induced electron flow is indicated below
The observed shifts can be either upﬁeld or downﬁeld. Fig:Left: Anisotropy from a benzene π system.LECTURE COURSE: NMR SPECTROSCOPY
must be sterically ﬁxed in relative to the anisotropic group. The following ﬁgure shows regions of increasing (+) or decreasing () ﬁeld:
+ +
FIGURE 7. one can sometimes judge from the signal dispersion of 1D proton spectra whether a protein is folded or random coil.C + C 
These effects can be dramatically. Right: Anisotropy from a triple bond. Chemical shift anisotropies of various πsystems


C C + 
+ + C C . This is the case for folded proteins that do adopt a unique structure. those in the plane to values higher than 10 ppm.
5 Electric ﬁeld gradients: Strongly polar groups create intramolecular electric ﬁelds. r the distance to the centre and χ the magnetic susceptibilities parallel and perpendicular to the bond axis. The effect is especially pronounced for symmetric hydrogen bonds (those in which the distance of the proton is equal to both acceptors).59
. Protons that are part of hydrogen bonds do exchange much more slowly with labile solvent deuterons and can therefore be differentiated from others. As can be seen from the formula.6 Hydrogen bonds: Hydrogen bonds decrease the electron density at the involved proton site and hence leads to a highfrequency shift. This has the effect of distorting electron density in the rest of the molecule and will hence inﬂuence the chemical shifts.
Fourth Chapter: Chemical shifts and scalar couplings
Pg. The hydroxyl group at the top of the molecule is part of a sym.LECTURE COURSE: NMR SPECTROSCOPY
The chemical shift anisotropy for a triple bond can be calculated as ∆σ = 1 ⁄ 3r [ ( χ II – χ ⊥ ) ( 3 cos θ ) ]1 ⁄ ( 4π ) where theta is the angle with respect to the bond axis. the effect depends strongly on the distance and orientation. 1. concentration and solvent dependent. This is used in protein NMR to identify βsheets or α−helices that display extended hydrogen bond networks.
3
O
H
O
HO
FIGURE 8. Protons that are hydrogen bonded usually be easily recognized from their shift. Hbond and will therefore be
downfield shifted. Their shift is highly temperature. 1.
since electron density of the unpaired electron at the observed nucleus (which is usually not in the same molecule) is required. a 2 ppm shift range for aliphatic protons can be dispersed over 6 ppm after addition of the shift reagent. the chemical shift can also be inﬂuenced.60
. 1. Suitable solvents for causing large changes in chemical shifts are benzene or acetone.7 Solvent effects: It is very important to realize that different solvents may have different effects on the chemical shifts. This orientation rapidly changes in solution due to molecular tumbling and the dipolar coupling there
Fourth Chapter: Chemical shifts and scalar couplings
Pg. Quite often an overlapping signal can be resolved when changing the solvent.
2. which may completely change the appearance of a spectrum that has been acquired in chloroform. This effect is moderate. Thereby. This effect is especially pronounced when benzene selectively complexes parts of the molecule due to the ringcurrent effect from the aromatic ring. The mechanism is Fermicontact interaction (see section scalar couplings) between the nuclear spin and the unpaired electron.proton coupling is removed rapidly (selfdecoupling). This is so because the dipolar coupling depends on the orientation of the connecting vector to the static ﬁeld.8 Shifts due to paramagnetic species: Paramagnetic compounds have unpaired electrons. also known as shift reagents.LECTURE COURSE: NMR SPECTROSCOPY
1. Paramagnetic reagents. serve to disperse proton spectra. Since relaxation times of electrons are very short. SCALAR COUPLINGS:
Two types of interaction between spins are known: • • dipolar coupling scalar coupling
The contributions from dipolar coupling usually cannot be observed in isotropic (liquid) phase. the lines are usually considerably broadened for solvent excessible protons. When paramagnetic impurities are contained in the sample. Such selective effects may be the case when not all parts of the molecule can be accessed by the solvent. the splitting due to the electron. so that comparably sharp lines can be observed. However.
it can be observed in the solid state or in liquid crystals.or β. then the two sorbitals of the separate atoms form a σorbital. The scalar coupling leads to a splitting of resonance lines. Is the atom bonded to another atom.LECTURE COURSE: NMR SPECTROSCOPY
fore averages to zero. Spin states of nuclear (red) and electron (blue) spins for hydrogen atom (a) and singlet hydrogen molecule (b) and triplet hydrogen (c) with corresponding energy diagram. ΨA(0)2 is the electron density at the site of the nucleus A and cA the coefﬁcient of the contribution of the atomic orbital A to the σ MO. The magnitude of the coupling is expressed as the scalar coupling constant and can be calculated as: J = ( 2µ 0 g e µ B ⁄ 3 ) γ A γ B Ψ A ( 0 ) Ψ B ( 0 ) c A c B ( 1 ⁄ ∆ T ) where γA and γB are the gyromagnetic ratios of the involved nulcei. which is occupied by two electrons with antiparallel spin (Pauli principle).state. ∆T is the triplet excitation
2 2 2 2 2
Fourth Chapter: Chemical shifts and scalar couplings
Pg. The second nuclear spin can now have its spin parallel or antiparallel to the spin of the second electron depending on whether it is in the α.
Electrons that occupy an orbital that has a nonvanishing electron density at the nucleus (such as selectrons) have the electron spin antiparallel to the nuclear spin (Hund's rule).61
. This effect describes the coupling between the nuclear and the electron spin:
E
b c a a c b
∆E= hν = hJ
FIGURE 9. The basic mechanism that propagates the coupling is the Fermicontact interaction. However. The effect is mediated via the electrons and its magnitude therefore rapidly decreases when the number of intervening bonds increases. The antiparalell alignment is energetically favourable and hence gives a signal at lower frequency.
The scharacter of orbitals depends heavily on the hybridization of the involved nuclei. which are deﬁned as: κ ik = ( J ik ⁄ h ) ( 2π ⁄ γ i ) ( 2π ⁄ γ k )
2. the magnitude of coupling depends on the amount of overlap of the involved electron orbitals.13C coupling constants which can be roughly categorized as: •sp3: 140 Hz •sp2: 160Hz •sp: 250 Hz The sp orbital has the highest content of scharacter and hence gives the largest coupling.
Fourth Chapter: Chemical shifts and scalar couplings
Pg.62
. meaning that they don’t have the same chemical shift.1 Direct couplings (1J): In order to be able to detect scalar spin.spin couplings it is necessary that the two protons are not isochronous. As we will see later. The onebond coupling constant usually has a positive sign. When hydrogen is substituted by deuterium.LECTURE COURSE: NMR SPECTROSCOPY
energy that measures the energy required to promote an electron from the singlet into the triplet spin state (parallel alignment):
∆T
FIGURE 10. The coupling also depends on the gyromagnetic ratios of the involved nuclei. in which n indicates the number of intervening bonds.514 . Singlettriplet transition
The scalar coupling constant is usually given the symbol nJ. Therefore. the couplings are sometimes reported in form of the reduced couplings. the corresponding coupling to another proton is scaled by the factor γD/γH=1/6. This is very clear from the 1J 1H.
2 Geminal couplings (2J): Geminal proton couplings largely depend on the hybridization of the involved carbon nuclei:
H2C
H 109 H
H 120 H . it
Fourth Chapter: Chemical shifts and scalar couplings
Pg.9 cosφ + 3.3 Hz
H2C=C
H 120 H
12.81 .5 Hz
To be able to observe these couplings the geminal bonded protons need to be diastereotopic in order to give separate proton frequencies.83 cos2φ
This relationship. Of course.3 Vicinal couplings (3J): Vicinal couplings display a characteristic dependence upon the involved dihedral angle:
3J (Hz)
H
b
φ
H
a
φ/rad
FIGURE 12.4 Hz
FIGURE 11. 2. Various geminal couplings
+2. For 1H.0 cosφ + 5 cos2φ
for protons. has been successfully exploited to determine the stereochemistry of compounds.4.LECTURE COURSE: NMR SPECTROSCOPY
2.1.63
. also known as the Karplus relation. Left: Karplus curve.0.13C couplings a similar equation has been suggested:
3J=
3. right: Definition of dihedral angles
according to the relation:
3J=
7 .
the coupling is very useful to determine the stereochemistry or derive structural constraints:
H1a
R Ha Hb 3 Ja.b ≈ 11 Hz R' Ha
R Hb R' 3 Ja . gauche and trans rotamers.b ≈ 1819 Hz
H2e H2a 3 Ja.64
.g. Overlap of adjacent orbiatals for φ=0° (left) and φ=90° (right). in sugars the vicinal coupling between the anomeric proton and the neigbouring proton determines whether the sugar is α or β. because the dihedral angle determines how large the overlap of the molecular orbitals and thereby the efﬁciency of transfer is:
φ
FIGURE 14.e = 25 Hz 3 Ja.LECTURE COURSE: NMR SPECTROSCOPY
requires the dihedral angle to assume a certain value ﬁxed over time. The reason for the dependence of the vicinal coupling on the dihedral angle is the follwoing: The scalar coupling is propagated via electrons. For systems where the bond cannot be rotated (e. e. which for that case corresponds to the arithmetic mean from the mainly populated rotational states. e = 25 Hz
FIGURE 13. Vicinal couplings of systems with hindered rotation about CC bonds
For example. rapid averaging of dihedral angles (rotatable bonds) leads to averaging of coupling constants. Openchain aliphatic compounds therefore mostly display vicinal couplings of about 7 Hz.g.
Fourth Chapter: Chemical shifts and scalar couplings Pg. a = 1013 Hz 3 Je. Therefore. The angular dependence comes into play. cyclic systems or macromolecules with deﬁned secondary structure such as proteins).
π electrons cannot propagate scalar couplings because π orbitals have nodes at the position of the nuclei.π transfers
Since π electron systems are highly delocalized. these interactions can be propagated over many bonds.6 The number of lines due to scalar spin.4 Longrange couplings:
4J
couplings are rarely observed. when couplings are of similar magnitude. e. Systems with 4J couplings. each coupling doubles the numbers of lines. Therefore. The orientation of the double bond in 4J couplings is not important.5 Couplings involving π electrons: In principle. However. some lines overlap. 2.g.
2. cisoid and transoid couplings are of similar magnitude. In the absence of geminal couplings (no diastereotopic protons) all couplings are usually due to vicinal
Fourth Chapter: Chemical shifts and scalar couplings Pg. For saturated systems they require the atoms
to be coplanar and in zigzag (W) conformation:
H
H4e
H2 e
H
FIGURE 15.spin couplings: In principle. spincorrelation between σ and π electrons is required:
π
H C
σ
H H
H H H H
C
FIGURE 16. Scalar couplings involving σ.LECTURE COURSE: NMR SPECTROSCOPY
Further effects on the vicinal couplings are: • • • electron negative substituents decrease 3J increasing HCC bond angles decrease 3J increasing CC bond lengths decrease 3J
2.65
.
LECTURE COURSE: NMR SPECTROSCOPY
protons.
The intensities of the lines in the case of overlapping lines (similar magnitude of couplings) can be derived from the coefﬁcient in the Pascal triangle:
Number of coupled nuclei I=1/2 I=1 Number of lines rel.
Jac Jab Jac J ab
J ac
Jac
Ωa
Ωa
FIGURE 17. Coupling of a proton a with two other protons b and c for the case of different (left) or
similar (right) couplings.66
. In this case the total number of lines of a single resonance due to the coupling with N neighbouring protons is N+1. In openchain compounds single bond dihedral angles are rotationally averaged so that all vicinal couplings are around 7 Hz (vide supra)´. intensities
0 1 2 3 4 5 6 0 1 2 3
1 (singlet) 2 (doublet) 3 (triplet) 4 (quartet) 5 (quintet) 6 (sextet) 7 (septet) 1 (singlet) 3 (triplet) 5 (quintet) 7 (septet)
1 1:1 1:2:1 1:3:3:1 1:4:6:4:1 1:5:10:10:5:1 1:6:15:20:15:6:1 1 1:1:1 1:2:3:2:1 1:3:6:7:6:3:1
Fourth Chapter: Chemical shifts and scalar couplings
Pg. In cyclic or structurally welldeﬁned compounds where the couplings may be much different the total number is 2(N1) .
these rules do not apply anymore.9 4. For the case of two dublets due to two strongly coupled protons the inner lines are larger then the outer ones ("roof effect"). Second order effects depending on the chemical shift difference. JBX =4Hz)
1
2
3
4
5. when the chemical shift difference is not very large compared to the scalar coupling constant (∆δ> 10 J). 3(2).8 ppm 5.
AB−System
(JAB =10Hz)
ABX−System
(JAB =10Hz.0 ppm
FIGURE 18. 1(3) and 0(4). However. JAX =6Hz.67
.0 4. The simulations were calculated using νA−νB/J=15(1).0 4. A practical consequence is that a resonance line cannot be referred to belong to a spin A or B.9 4. Thereby it is possible to decide which signals are coupled with each other.
Fourth Chapter: Chemical shifts and scalar couplings
Pg. In the case of the ABX system the Xpart is shown separately. Whereas in the limit of weak coupling a spinﬂip of spin A does not cause spin B to ﬂip there is a probability to do so in the strong coupling case. In the strongcoupling case spin properties are mixed.7 Strong coupling: The rules for line multiplicities and intensities described above are only valid in the case of weak coupling.8 ppm 4. In order to derive coupling constants or chemical shifts from strongly coupled spins these parameters may not be extracted from the spectrum but have to be derived from a simulation and comparison to the measured spectrum.LECTURE COURSE: NMR SPECTROSCOPY
2.
68
= e
− ( Eα − E β ) / kT
. Phenomenologically. 2.or the βstate. The process that brings the spins back to thermal equilibrium is called relaxation:
Bo=z
Bo=z y x
y x
FIGURE 1. The corresponding time constant of that process is called T1. However. phases are correlated
It is important to note that a particular spin can be only in either the α. the corresponding time constant is called T2.LECTURE COURSE: NMR SPECTROSCOPY
1. What relaxation describes is the behaviour of magnetization which is deﬁned as the sum of the magnetic moments from all spins. the spins are uncorrelated in phase such that no transverse magnetization exists. right: nonequilibrium state. to understand the mechanisms of relaxation we will look at the fate of individual spins. relaxation is categorized into 1. longitudinal relaxation (or T1 relaxation) that describes the return of the zcomponent of longitudinal (z)magnetization to its equilibrium value. Analogously. transverse relaxation (or T2 relaxation) that describes the decay of transverse (x.and βstates corresponds to the Boltzmann distribution. Furthermore. However.1 T1 relaxation: The equilibrium distribution of α vs. β state is governed by the Boltzman distribution: # (α ) # (β )
Sixth Chapter:Relaxation Pg. when RF pulses are applied the state of the spins is pertubated away from equilibrium distributions. RELAXATION:
As stated in the ﬁrst chapter thermal equilibrium is a state in which the population of α.y) magnetization. left: equilibrium state. 1.
Return of the zcomponent of magnetization to the equilibrium value.5 to a few seconds.
Note that the vectors shown in the ﬁgure above represent the magnetization vectors rather than magnetic moments from individual spins. Therefore.
1. T1 relaxation is an enthalpic process.spin relaxation:
Sixth Chapter:Relaxation
Pg. The surrounding of spins is often called the lattice and hence T1 relaxation is often referred to as spinlattice relaxation:
z y x
z y x
z y x
z y x
z y x
FIGURE 2. the delay that has to be inserted between individual scans.2 T2 relaxation: The transverse relaxation determines how fast phase coherence between spins is lost. Degassing the sample will remove paramagnetic oxygen which otherwise facilitates T1 relaxation.LECTURE COURSE: NMR SPECTROSCOPY
Since α and β states correspond to different energies changing the relative populations of these states changes the energy of the system. T1 times for protons are in the range of 0.69
. T1 times for quadrupolar nuclei (I > 1/2) can be rather short (ms range). degassing samples is useful when measuring small NOE effects. it is not changing the energy of the system. T2 relaxation is merely a entropic process. Therefore. Since dephasing in the spins does not cause any changes in the relative population of α− and βstate. The energy is transferred to or taken from other spins. It is often referred to as spin. Longitudinal magnetization decays exponentially according to Mz (t ) = M z (t0 ) e − t / T 1
The T1 relaxation time determines the pulse repetition rate. The initial state may
have been created through population inversion arising from an 180˚ pulse.
For example.to the βstate or vice versa.3 The mechanisms of relaxation: In principle the spin system may return to equilibrium via stimulated relaxation (some kind of energy exchange process that initiates relaxation) or by spontaneous relaxation. relaxation is caused by additional ﬁelds that happen to have exactly the frequency that corresponds to the required transition.t/T
2
t
t
FIGURE 3. Top: Dephasing of transverse coherence leading to T2 relaxation. The linewidth at half height of the signals can be expressed as:
∆ν 1 / 2 = 1 πT2
There is no reason to sample the signal for times much longer than T2 because only noise will then be added to the FID. T1 relaxation takes place if spins change from the α. The shorter T2 the broader the signals.70
.
Sixth Chapter:Relaxation
Pg. Mechanistically.LECTURE COURSE: NMR SPECTROSCOPY
X
X
X
X
X
X
y
y
y ∆t
y
y
y
I Det
I Det e. y (t ) = M x . y (t0 ) e − t / T 2
The transverse relaxation time determines the linewidths of the signals. Lower: Influence of T2
relaxation on the appearance of the FID.
Once the transverse relaxation time T2 passed by transverse magnetization has decayed to 1/e of its original value: M x . The T2 time and hence the linewidth also determines the theoretically achievable resolution. It can be shown that the probability for spontaneous relaxation is far too low to account for it. 1.
Right: Overall tumbling changes φ. Through molecule tumbling the angle φ permanently changes its magnitude causing a ﬂuctuating ﬁeld.dipole interaction with other protons the transverse relaxation time T2 and thereby its linewidth is determined by a) how far the next protons are in space and b) the overall tumbling time of the molecule.
The induced local ﬁeld can be expressed as: Bloc =
µ (3 cos2 φ − 1) r3
The magnitude of the induced ﬁeld depends on both the distance of the two interacting nuclei and on the angle formed between the interatomic vector and the static ﬁeld. It is often found that isolated protons (those that do not have other protons on the neighbouring carbons) display incorrect integrals in 1D proton spectra. For protons that relax mostly through dipole. Left: definition of the vector connecting two spins and its orientation φ with respect to the external field. This is due to the fact that T1 relaxation for these protons is
Sixth Chapter:Relaxation
Pg.71
.LECTURE COURSE: NMR SPECTROSCOPY
The energy difference between these states correspond to a speciﬁc frequency ∆E = Eα − Eβ = hν
and this frequency matches to a ﬁeld ﬂuctuating at
ν =
γB 2π
There are two major mechanisms by which these ﬂuctuating ﬁelds are formed: • • dipoledipole interaction between two different spins chemical shift anisotropy
Both processes are connected to the movement of spins in space due to overall tumbling of the molecules or due to internal motions. Dipoledipole induced relaxation: Spin A close in space to spin B causes a (small) dipole moment at the nucleus B:
Bo
1H
Bo
r
φ
15N
φH
N
FIGURE 4.
1.72
. 1.spin coupling to another nucleus ﬂuctuates rapidly. Dependence of electron shielding for carbonyl carbons on the orientation of the external field relative to the pz orbital
The efﬁciency of CSA relaxation increases with the square of the magnetic ﬁeld and is the major relaxation source for amide nitrogen nuclei at ﬁelds higher than 600 MHz. a method that depends on relaxation properties of spins and helps to elucidate the threedimensional arrangement of protons (see next chapter). occurring for sp2 hybridized carbon nuclei) will cause a ﬂuctuating ﬁeld upon rotation of the nucleus which serves as a efﬁcient source for relaxation:
σzz σzz
C
O
C
O
FIGURE 5. The linebroadening of signals due to slow overall tumbling is obvious from the linewidth of spectra from large proteins that display very broad lines. The distance between geminal protons depends on the bond geometries but for all other protons it is determined by the structure.4.4 Other relaxation mechanisms: Any particular event that will cause a ﬂuctuating ﬁeld at the site of the nucleus may contribute to relaxation provided that it delivers spectral density at any of the possible NMR transitions. 1. This could be caused by • chemical exchange of the coupled nucleus (scalar relaxation of the first kind)
Sixth Chapter:Relaxation
Pg. An important application therefore is the NOE effect.LECTURE COURSE: NMR SPECTROSCOPY
inefﬁcient and therefore these protons do not have completely relaxed in between different scans.4. It is also the major relaxation source for nonproton bearing carbon nuclei such as carbonyl or oleﬁnic carbons.2 Scalar relaxation: Relaxation may also be induced when the scalar spin.g.1 Chemical shift anisotropy (CSA): Nonspherical distribution of electron density (e.
The effects are dramatic such that small contaminations with paramagnetic impurities may severely broaden the lines. 1. little (middle) or
Sixth Chapter:Relaxation Pg.4.5 Interaction with unpaired electrons: The interactions can be either of dipolar or scalar nature. Therefore. The timedependence of the motions is of prime importance since it determines the frequency at which the induced local ﬁelds ﬂuctuate and therefore whether they may contribute to relaxation or not. Scalar relaxation of the second kind is frequently encountered for protons coupled to quadrupolar nuclei.
Scalar relaxation of the ﬁrst kind is observed for protons coupled to quickly exchanging hydroxyl protons. This effect is usually of minor importance. T1 and T2 relaxation is very fast for these nuclei provided that the ligand environment of the nucleus is nonsymmetrical. The following ﬁgure displays the correlation function for functions with no (left).diradical) is often removed through thaw and freeze cycles to increase T1. 1.LECTURE COURSE: NMR SPECTROSCOPY
• rapid T1 relaxation of the coupled nucleus (scalar relaxation of the second kind). An practical example is the case of nitrile carbons in which the carbon is mostly coupled to the quadrupolar 14N nucleus and is usually signiﬁcantly broadened.4.4. In order to describe how the angle φ changes with time we use the correlation function g(τ): g(τ ) = f (t ) f (t + τ )
that describes how quickly a function changes with time. carbonyl nuclei may have T1's of 2030sec) to allow faster pulsing. Scalar relaxation is much more likely to effect T2 than T1 relaxation times.73
. oxygen (which is paramagnetic. which gives a narrow resonance in the tetrahedral NH4+ ion but much broader lines in asymmetrical environment such as pyrrole. 1. 1. This is seen for 14N.4 Spinrotation relaxation: Rapidly rotating groups such as methyl groups induce a ﬂuctuating ﬁeld at the frequency of rotation.3 Quadrupolar relaxation: Nuclei with a spin > 1/2 rapidly relax under the inﬂuence of a electric ﬁeld gradient. For 13C NMR relaxation reagents like Cr(acac)3 are added in very small quantities to bring the T1's down to about 1s (otherwise.5 The motional properties: In this section we will see how the dependence on the motional properties is expressed mathematically.
LECTURE COURSE: NMR SPECTROSCOPY
rapid (right) time dependence:
FIGURE 6. We are not interested so much into the time it takes for a molecule to change its spatial arrangement but rather on the frequency with which these changes take place. However. smaller molecules may rotate faster than larger molecules so that the highest possible frequency is lower for larger molecules. This can be rationalized in the following way: The overall power from all frequencies (the integral under the spectral density function) must remain the same for all molecules. shape etc. are preferred.
Since the NMR signal decays exponentially (this can be derived from the lineshape which must be lorentzian for exponential decay). slow decay (middle) and fast decay (right. A very approximate formula valid for globular proteins in water is τc ≈1012 M (M = molecular weight in Dalton) but depends highly on temperature. It is clear that the densities at low frequencies increase with increasing correlation times (larger molecules). typical correlation functions for no decay (left).74
. g(τ) is believed to be exponential:
g(τ ) =
e −τ / τ c
τc is the correlation time which is deﬁned to be the time required for a 360˚
rotation of the molecule. Therefore. It can be calculated as:
J (ω ) = 2τ c 1 + ω 2τ c 2
The dependence of the spectral densities on the frequencies are shown in the ﬁgure below for large (left).
Sixth Chapter:Relaxation
Pg. the spectral densities. mediumsized (middle) and small molecules (right). which are the Fourier transform of the correlation function. The spectral density J(ω) tells us how much power is available from the motion of the molecule to cause ﬂuctuations at the frequency ω.
1 The dependence of the relaxation rates on the fluctuating fields in x.
Sixth Chapter:Relaxation
Pg. ﬁelds in x. We will see later that the frequencies that match possible NMR transitions determine how quickly a nucleus may relax. Then it hits another molecule and the rotation is slowed down or stopped. Spectral density functions for no decay (left).In contrast.
Small molecules therefore posses a broad band of rotational frequencies whereas the larger molecules have more spectral density at the lower frequencies (in fact. medium decay (middle) and fast decay (right). the highest spectral density is at frequency zero).y or z direction: An additional ﬁeld in zdirection means that the spins additionally precess about the zaxis with the frequency of that ﬁeld.5.or ydirections cause spins to ﬂip from the αto the βstate or vice versa.
The motional fate of a molecule can be described by the following scenario: A molecule rotates with a certain frequency. After the crash the molecule starts to rotate again until it has reached its highest possible frequency or has hit another molecule:
H H N H N N N N H H
ν
ν
ν
ν
FIGURE 8. 1.75
.LECTURE COURSE: NMR SPECTROSCOPY
FIGURE 7.
rotations about the x. The transverse relaxation time is determined by the amount of dephasing of transverse magnetization. Obviously. In addition. This can be seen from the Heisenberg principle which states that the shorter the lifetime is the larger the uncertainty in energy is.to βstate transitions and therefore on the ﬁelds along x or y:
1 T1
2 2 ∝ γ 2 ( H x + H y ) J (ω )
In order to be effective these ﬁelds must precess with frequencies comparable to the larmor frequency about z.76
.LECTURE COURSE: NMR SPECTROSCOPY
Bo=z
y x
FIGURE 9. say from the +zdirection to the zdirection.
Sixth Chapter:Relaxation Pg. ﬁelds along z change the precession frequency since they alter the strength of the static ﬁeld (which is also along z):
1 T2. This is analogous to the case of the B1 ﬁeld which can only inﬂuence spins when the ﬁeld precesses about the zaxis. x 1 T2. Dephasing at J(0) is effective because if the zﬁeld would ﬂuctuate.to βtransitions) will reduce the lifetime of the state (lifetime broadening). It can therefore only be effective if that ﬁeld stays in the +z direction and hence the spectral density at zero frequency is important. The contribution of lifetime broadening to T2 is the same as to T1 since it uses the same mechanism and hence similar spectral densities are required. y
2 2 = γ 2 ( Hz J (0) + H y J (ω ))
2 2 = γ 2 ( Hz J (0) + H x J (ω ))
This mechanism is represented by the ﬁeld term along z in the equations above.
The longitudinal relaxation time depends on the frequency of α.or yaxis (causing α. the dephasing effect from it would be reversed and averaged out.
77
. τe is the effective correlation time for that internal motion. The spectral densities in this model is expressed as:
J ( ω ) = S ( τ R ⁄ ( 1 + ωτ R ) ) + ( 1 – S )τ e ⁄ ( 1 + ωτ e )
2
2
2
2
The strength of the LipariSzabo interpretation is that it gives an impression about the relative “ﬂoppiness” of parts of the molecule in absence of any speciﬁc model about the exact nature of the internal motion.LECTURE COURSE: NMR SPECTROSCOPY
1. Assuming free diffusion in a cone the halfangle θ expressing the spatial restriction of the internal motion can be calculated as
S ( θ max ) = 0.5 cos ( θ max ) ( 1 + cos θ max )
which translates for an S2 of 0.6 Excurs: The LipariSzabo model for motions:
The LipariSzabo model (also sometimes referred to as the “modelfree” approach) factorizes the autocorrelation function into a correlation function for overall tumbling and one for internal motions:
g ( t ) = go ( t ) • gi ( t )
The overall correlation is assumed to decay exponentially
–t ⁄ τR
go ( t ) = e
The correlation function describing internal motions is described as
g i ( t ) = S + ( 1 – S )e
2
2
–t ⁄ τe
The generalized order parameter S2 describes the spatial restriction of the internal motion with respect to a moleculeﬁxed coordinate system.4 into a half angle of 45°:
2
2
Sixth Chapter:Relaxation
Pg. A value of 1 means that a particular vetor does not move relative to the molecular frame and a value of zero indicates that movement of a vector is completely uncorrelated to overall tumbling which is the case for highly ﬂexible loop regions in globular proteins for example.
to the αstate in a correlated fashion (zeroquantum transition).to the βstate (doublequantum transition).78
.to the βstate is called a singlequantum transition:
ββ
ββ
βα
αβ
βα
αβ
αα
αα
i Spin single quantum transitions
SSpin
FIGURE 11. For homonuclear spin systems the zeroquantum frequencies are very low (kHz range) and the double quantum frequencies very high. As we have seen before J(0) steadily increases with increasing molecular size and for larger molecules such as proteins almost all spectral density is at J(0).to the βstate and another one from the β.
Sixth Chapter:Relaxation Pg. A transition that involves a single spin going from the α. left: Ispin single quantum transitions.LECTURE COURSE: NMR SPECTROSCOPY
1.
ββ
ββ
βα
αβ
βα
αβ
αα
αα
zero quantum transition
FIGURE 12. Furthermore one spin may go from the α. It can be seen from the ﬁgure below that for T2 relaxation spectral density at J(0) is important in contrast to T1 and NOE. Right: Sspin single quantum transitions
Alternatively two spins may simultaneously go from the α. zero. Hence T2 times are very short for large molecules leading to very broad lines.7 The nature of the transitions: So far we have only been talking about a single spin changing his state due to interaction with the surrounding spins. The frequency for the double quantum transition corresponds to the sum of the frequencies of the individual spins whereas the zeroquantum frequencies are formed from the difference of their corresponding frequencies.and double quantum transitions
double quantum transition
For T1 and T2 processes spectral densities at frequencies corresponding to different transitions are utilized.
single quantum nitrogen coherences and heteronuclear (15N.
For 15N magnetization. T2 and NOE upon spectral densities at various frequencies. Fore large molecules. For 1H relaxation. and this is the principal reason why large molecules display very broad nmr lines. the zero quantum frequencies (which are the difference of the chemical shifts) are in the 010 KHz range and therefore very close J(0) whereas for 15N nuclei the zeroquantum frequencies are not so much away from the proton frequencies (MHz range). that the rates of T1 and T2 relaxation differ in the contribution of J(0) for T2.79
ω N + ωH
ωN
o
.1H) double and zeroquantum coherences:
1 = R N (N Z ) = T1
2 2 γ H γ N h2 J ( ω H − ω N ) + 3 J ( ω N ) + 6 4r N . Hence. Dependence of T1.LECTURE COURSE: NMR SPECTROSCOPY
Note that the parameters in the ﬁgure below are given for 15N relaxation data (the mechanism is dipolar relaxation with 1H).
ω N . T2 relaxation is very fast.H 6 J ( ω H + ω N )
+
2 ∆2 ω N J (ω N ) 3
1 = R N (N x .ωH
ωH
T1
T2 NOE
FIGURE 13. Note.y ) = T2
2 2 γ H γ N h2 4 J (0 ) + J ( ω H − ω N ) + 3 J ( ω N ) + 8r 6 .
Sixth Chapter:Relaxation
Pg. relaxation is mainly due to 1H. J(0) contains almost all spectral density.H 6 J ( ω H ) + 6 J ( ω H + ω N ) N 2 ∆2 ω N 2 1 J (0 ) + J ( ω N ) 3 3 2
+
in which ∆ denotes the chemical shift anisotropy of nitrogen.15N dipolar interactions and 15N chemical shift anisotropy. It depends on spectral density of single quantum proton coherences.
8 Measurement of relaxation times: T1 relaxation times are usually determined from the inversion recovery sequence: 180 deg pulse…delay…90 deg pulse…delay .. Dependence of T1 and T2 upon the overall tumbling time
1. T2 continuously gets shorter with increasing molecular size due to the increase in spectral density at J(0).acquisition
Sixth Chapter:Relaxation
Pg.80
. Following the point where spectral density at the zeroquantum frequencies decreases T1 is getting larger again. (T1 relaxation does not depend upon spectral density at J(0):
T1
T2
FIGURE 14. T1 however passes a minimum.LECTURE COURSE: NMR SPECTROSCOPY
The dependence of transverse and longitudinal relaxation upon the correlation time is shown in the next ﬁgure.
However. The magnitude and sign of the xmagnetization at the end of the sequence depends on a) the T1 constant and b) the relaxation delay ∆. one could simply measure the decay of transverse magnetization following a 90 degree pulse. magnetization is inverted by application of a 180 degree pulse. Afterwards a 90 degree pulse along y turns the magnetization onto the xaxis into observable signal. A delay follows during which T1 relaxation takes place bringing the z magnetization back towards +z. Inversion recovery. Usually.
In this sequence. In principle.LECTURE COURSE: NMR SPECTROSCOPY
z y x
180 deg
z y ∆t x
z
90 deg
z
z y x
90 deg
z
2∆t
FIGURE 15. one is not interested into the contribution from ﬁeld inhomogeneity (this would mean that the transverse relaxation time would depend on the quality of shimming) and therefore the CarrPurcell sequence that eliminates the additional T2 relaxation due to ﬁeld inhomogeneity is applied:
Sixth Chapter:Relaxation
Pg.81
. Transverse relaxation time is determined from a CarrPurcell Spin echo. inhomogeneity of the static ﬁeld leads to accelerated transverse relaxation.
82
. The effect of the 180˚ pulses is to refocus differential precession due to ﬁeld inhomogeneity. CarrPurcell spin echo. For both experiments. the inversion recovery and the CarrPurcell sequence a set of 1D (or 2D) spectra is recorded with different relaxation periods. The intensities of the remaining signal is measured and plotted against the relaxation delay. The sequence is also referred to as an spinecho. Mostly these plots yield decaying exponentials and by ﬁtting procedures (least squares ﬁt to the experimental data) it is possible to extract the time constant for the decay:
Sixth Chapter:Relaxation
Pg.
In this sequence 180 degree pulses are applied during the T2 relaxation delay equally spaced by delay periods.LECTURE COURSE: NMR SPECTROSCOPY
z y x
90 deg
z
∆t
z y x
z
z y x
180 deg
z y x
∆t
FIGURE 16.
2
1.LECTURE COURSE: NMR SPECTROSCOPY
T1
Intensity Intensity
T2
[sec] 0. Analytical formula to describe the decay of longitudinal (left) and transversal (right) magnetization.4
0.8
1.83
.6
2.
Sixth Chapter:Relaxation
Pg.0
[msec]
80
160
240
I(t) = A e R1/t + B
I(t) = A e R2/t + B
FIGURE 17.
The NOE effect is the method for elucidation of 3D structural features and stereochemistry using NMR together with information from scalar spin. the NOE is the major tool for elucidation of stereochemistry of molecules in solution. The NOE depends on the relaxation rates of zero. respectively. it characterizes the rate of
Seventh Chapter: The NOE Effect Pg. THE NUCLEAR OVERHAUSER EFFECT (NOE):
If one resonance A is irradiated.84
.and double quantum transitions:
fI {S} =
γ S σ IS γ I ρ IS
σIS is the crossrelaxation rate.LECTURE COURSE: NMR SPECTROSCOPY
1. Physically speaking.spin couplings. an increase (positive NOE) or decrease (negative NOE) of signal intensity of other resonances such as resonance C is observed when spin C is close in space to spin A:
A
B
C
Reference
positive NOE
A
C
B
negative NOE
FIGURE 1.
This phenomenon is called nuclear Overhauser effect or NOE. Irradiation of resonance A leads to a increase of peak intensity of the neighboring spin C (positive NOE) or to a decrease of peak intensity (negative NOE). The NOE enhancement factor η is deﬁned as
η =
fI {S} =
(I − I )
o
Io
in which I and Io is the observed intensity of the resonance of spin I with and without irradiation of spin S. The efﬁciency through which the NOE is transferred from spin I to spin S depends strongly on the distance of the two protons involved and on the tumbling properties of the molecule:
η = f (τ c r −6 )
Because of the distance dependence.
It corresponds to the rate of recovery of Isignal after selective inversion. In a twospin system is ρIS equals to 1/T1. however. Dependence of the strength of the NOE on spin A caused by irradiation of spin B on the bond angle α. The important point to note here is that the magnitude of a NOE between two particular spins not only depends on their distance (and on the tumbling of the molecule) but also on the fact how many other protons are in close distance:
fA{B}
α
FIGURE 2. It is the rate constant for direct dipolar relaxation of spin I by spin S.85
. In multispin systems.
This is nicely demonstrated in the ﬁgure above which shows the magnitude of the NOE between two protons A and B.LECTURE COURSE: NMR SPECTROSCOPY
dipoledipole transitions that give rise to the NOE. In the case of multispin systems R1 = 1 = ρ IS + ∑ ρ IX + ρ I* T1 x
ρIX corresponds to dipolar interaction with all other spins X and ρ* to other relaxation mechanisms for spin I such as CSA. ρIS is the longitudinalrelaxation rate. W2QC and WZQC are the rates for doublequantum and zeroquantum transitions. It can be expressed as:
σ IS
= W2 QC − WZQC
Therein. dipolar relaxation with other spins contributes to 1/T1 and then ρIS is not equal to 1/T1. Note that the distance rAB remained unchanged!
Seventh Chapter: The NOE Effect
Pg. The smaller the angle α the closer the third spin C is and the more efﬁcient dipolar relaxation of spin A by spin C becomes thereby dramatically reducing the magnitude of the NOE between spins A and B.
Utilizing these two different means of disturbing the populations of spin systems the experiments for measuring NOEs can be categorized into the following • the steadystate NOE experiment. A reference spectrum is recorded without irradiation and subtracted. After a delay τm for NOE buildup a 90˚ read pulse is applied and a 1D acquired.1 Experiments to measure NOEs: In principle.5s). This can be accomplished by • an inversion pulse (180 degree pulse) that inverts the relative populations of α. The famous NOESY (or ROESY) is the 2D variant.and β.and βstates to be equally populated. in which a resonance is selectively irradiated with low power for a time τm sufﬁciently long to completely saturate the transition and propagate the NOE (approx. the transient NOE. Again. which have changed their intensity) and the irradiated resonance. > 0. Middle: Population after selective Ispin inversion pulse.states saturation of a resonance line that causes the corresponding α. In this experiment a resonance is selectively inverted with a selective 180˚ pulse. Right:
Population after saturation of the Ispin resonance.86
. Left: Equilibrium population.
•
Seventh Chapter: The NOE Effect
Pg.
•
ββ
S
βα
I
αβ
S
I
S
I
I
αα
S
I
S
I
S
FIGURE 3.states of a spin system are different from the equilibrium value.and β.LECTURE COURSE: NMR SPECTROSCOPY
1. The difference spectrum shows only signals which have received a NOE (that means. one experiment with and one without the selective 180˚ pulse are subtracted from each other. a NOE enhancement can be triggered whenever the population of α.
The enhancement is:
− rIS6 − + ∑ rIX6 x
fI {S} = ηmax
− rIS6
−6 f X {S}rIX − ηmax ∑ −6 r + ∑ r −6 x IX IS x
In this equation the ﬁrst term represents the direct contribution to the NOE. In spindiffusion a relay spin that is close to both spins serves to transfer the NOE.
1. It is also called spindiffusion. The magnitude due to the direct NOE is inﬂuenced by nearby protons because they have an inﬂuence on T1. The second term in the equation above describes the indirect NOE transfer. Pulseschemes for measuring of steady state NOE (left) and transient NOE(right).2 The steadystate NOE: The steady state NOE depends on the geometry of all protons nearby. Depending on the correlation time and the sign of the NOE one distinguishes two cases: 1.87
• • •
Seventh Chapter: The NOE Effect
. not only on those saturated and observed.LECTURE COURSE: NMR SPECTROSCOPY
τm
NOE
τm
Ref steadystate tr ansient noe
FIGURE 4.
Pg.1 Extreme narrowing (ηmax >0): • Most enhancements are positive but some can also be negative.2. T1 and T2 values are very similar. The indirect transfer is a 3spin effect. depending on the geometry. This is shown in the ﬁgure below that displays the contribution due to the 3spin effect depending on the angle α (extreme narrowing). The lines are rather sharp (hence the name extremenarrowing). The inﬂuence of the indirect effect is smaller but noticeable.
2. The lines are broad.B.
f A{B}
NOE A 1 B 2 C 1 D
fC{B} f D{B} ωτC
FIGURE 6.LECTURE COURSE: NMR SPECTROSCOPY
f C{A}
total
direct
α
indirect
FIGURE 5.
1.2 Spindiffusion (ηmax <0): • • • In the negative NOE regime (large molecules). Steadystate enhancements depending on the correlation time for a fourspin system. The T2 values are very much shorter than T1.
Seventh Chapter: The NOE Effect Pg. Contributions of direct and indirect (spin diffusion) effects to the overall NOE. all enhancements are negative. Resonance B is irradiated. The
relative distances between spins A.88
.C and D are 1:2:1 as indicated in the insert.
the extreme narrowing and the spindiffusion limit. in which the irradiation time is limited to short periods. Spindiffusion can be recognized from the buildup curves of the NOEs (a number of NOESY experiments are recorded with increased mixing times. This is shown in the ﬁgure above.89
. When the molecules have gained a certain size the spindiffusion effect spreads the NOE out to all other protons (see the magnitude of the NOE for ωτc > 100) respectable of what their distance to the irradiated proton is! In order to circumvent the problems associated with spindiffusion the truncateddriven NOE has been used.
•
The following ﬁgure shows the transient NOE buildup upon irradiation of
Seventh Chapter: The NOE Effect Pg. • Using short mixing times NOE information is still useful in the spindiffusion case.LECTURE COURSE: NMR SPECTROSCOPY
Spindiffusion is very effective and steadystate NOE measurements are completely useless.3 The transient NOE: In the transient NOE experiment a resonance is inverted by application of a selective 180 degree pulse or unselectively in a 2D experiment (NOESY). 1D transient and NOESY experiments give identical enhancements. Spindiffusion cross peaks should show a characteristic induction phase). In both regimes. The NOE enhancement is: fI {S} =
σ − ( R' + D )τ m − e − ( R' − D )τ m ) (e D
R' = 1 ( RI + RS ) 2
D =
1 2 ( RI − RS )2 + σ IS 4
The transient NOE has some features that are remarkably different from the steadystate NOE: • Enhancements are symmetrical fI {S} = fS {I}
irrespective whether spin S has another proton close in space (which quenches the NOE in the steady state case dramatically). enhancements are nonsymmetrical due to possible relaxation leakage with a third spin: fI {S} ≠ fS {I}
1.
1. Offdiagonal elements are transitions. In thermal equilibrium all offdiagonal elements are zero. The density matrix contains diagonal elements which correspond to populations. In the spindiffusion limit it can still be distinguished from the genuine NOE peaks at short mixing times.90
.
In the extreme narrowing the spindiffusion peak β2H is of negligible intensity. the αβ. Lys56 β1H and β2H depending on the used mixing time for molecules in the extreme narrowing (τc = 3x1011 or 3x1010) or in the spindiffusion limit (τc = 3x109 or 3x108):
τC=3 x 10 11
NOE
τC=3 x 1010
τC =3 x 109
τC=3 x 108
FIGURE 7. A onespin system may be in the α or β state or may undergo a transition from the α into the β state (or vice versa). The
Seventh Chapter: The NOE Effect
Pg.LECTURE COURSE: NMR SPECTROSCOPY
Lys56 NH of bovine phospholipase A2 and observation of Ala55 NH (N). This is in contrast to the use of steadystate NOE's in that case. These are states that do not undergo transitions. A twospin system may be in the αα. the βα or the ββ state or may undergo transitions between them. The density matrix contains the probabilities for a given state or transition and hence is a convenient way to simultaneously represent all possible states of a spin system.4 The state of the spin system and the density matrix: The density matrix describes the state of the spinsystem. Strength of the spindiffusion peak (β2) in dependence of the correlation time.
LECTURE COURSE: NMR SPECTROSCOPY
αα → αβ or αα → βα αβ → αα
or βα → αα
or
transitions are called singlequantum coherences.
Seventh Chapter: The NOE Effect Pg. Occupied states of the density matrix (left) of populations and singlequantum transitions with corresponding transitions.91
. Double quantum transitions involve transitions where two spins ﬂip the same way and in zeroquantum transitions the two spins undergo opposite ﬂips. Double quantum coherences evolve with the sum of the chemical shifts of the comprising spins. The double quantum coherences:
αα → ββ or ββ → αα
and the zeroquantum coherences (ﬂipﬂop transitions):
αβ → βα
or βα → αβ
are not directly observable but play a very important role for the theoretical description of the nuclear Overhauser effect (NOE) because their efﬁciency determines the crossrelaxation rate. zero quantum coherences evolve with the difference of their chemical shifts:
αα αα αβ βα ββ αβ βα ββ
Populations
αα αα αβ βα ββ
αβ
βα
ββ
SingleQuantum Transitions
ββ ββ
βα
αβ
βα
αβ
αα ISpin SSpin
αα
FIGURE 8. Those are the only transitions which lead to observable signals during detection periods. since only one spin changes its state.
hence the change in the sign of the NOE:
Seventh Chapter: The NOE Effect
Pg.
1.LECTURE COURSE: NMR SPECTROSCOPY
αα αα αβ βα ββ
αβ
βα
ββ
ZeroQuantum Transitions
ββ
βα
αβ
αα
αα αα αβ βα ββ
αβ
βα
ββ
DoubleQuantum Transitions
ββ
βα
αβ
αα
FIGURE 9.and doublequantum transitions with corresponding transitions. spindiffusion limit).92
.proton NOEs the zeroquantum frequencies are close to zero and the doublequantum frequencies are very high. As we have seen before large molecules have spectral density only at frequencies close to zero. Occupied states of the density matrix (left) of zero. The dependence on the size of the molecule (to be more speciﬁc: on their correlation time) comes into play because the crossrelaxation rate is deﬁned as:
σ IS
= W2 QC − WZQC
For proton. extreme narrowing) or negative (large molecules.5 The sign of the NOE: The sign of the NOE can be positive (small molecules. Therefore. large molecules relax via zeroquantum transitions whereas small molecules mostly use doublequantum transitions.
is applied along a transverse axis (x. at RT).93
. the zero.or doublequantum transitions have much lower frequencies. why NOE spectra give very disappointing results for medium sized molecules (approx.and doublequantum transitions depends on the strength of the applied ﬁeld.
The absolute size of zero. In the rotating frame NOE (ROE) experiment an additional ﬁeld. the socalled spinlock ﬁeld. 2000 m. Spectral densities for a small (left) or large (right) molecule.
This is the reason.or yaxis).wt. At this size efﬁciency of zeroquantum and doublequantum transitions is approximately the same and therefore the crossrelaxation rate (which is the difference between the two rates) is close to zero:
FIGURE 11. NOE in
dependence of the correlation time. Because the B1 ﬁeld is much weaker (kHz range instead of MHz range). Middle: Max. In the ROE experiment magnetization therefore precesses about the x.(or y) axis with a much lower frequency as the magnetization precesses about the zaxis in the NOE experiment.LECTURE COURSE: NMR SPECTROSCOPY
J(ω)
J(ω )
og ω
og ω
FIGURE 10. NOE and ROE dependence on the correlation time. and hence relaxation always takes place via the
Seventh Chapter: The NOE Effect
Pg.
However the spins relax with T1ρ during the spinlock time (which is similar to T2) and hence the ROESY cannot be used for larger molecules due to rapid relaxation during the mixing time.5 times faster than the NOE buildup and hence shorter mixing times can be used. even for large molecules.
y x
ROE
The ROE buildup is about 2. In contrast. relaxation during the mixing time of a NOESY takes place with T1 which is long.94
.and doublequantum transitions contribute to the NOE In a twospin system the transitions α2>β2 give rise to the spin2 resonance and the transitions α1>β1 to the spin1 resonance:
β1β2 β1α2 α1α2
spin 2 tr ansiti ons
FIGURE 13. Axis of rotation during NOE (left) and ROE(right) buildup.6 Why only zero.
β1β2 α1β2 β1α2 α1α2
spin1 tr an sitions
α1β2
Let us assume that we irradiate the resonance of spin 2:
Seventh Chapter: The NOE Effect
Pg.LECTURE COURSE: NMR SPECTROSCOPY
doublequantum transitions and no zero crossing is observed:
Bo =z
Bo=z
y x B1 =y NOE
FIGURE 12. 1.
this system tries to go back to the equilibrium state. because irradiation of the spin 2 resonances still keeps the populations of the two α2 and β2 states equal leading to β1α2 = β1β2 and α1α2 = α1β2.
However.
Due to the irradiation the populations of the two states α2 and β2 are equalized.LECTURE COURSE: NMR SPECTROSCOPY
β 1β 2
2/ 4 2/ 4
β1β2
3/3 3/ 6
β 1α 2
4/ 8 4/8
α 1β2 α 1α 2
β1α2
3/ 6 6/ 6
α 1β 2 α1α 2
FIGURE 14. As can be seen from the following ﬁgure.or double quantum transitions::
αα → αβ or αα → βα αβ → αα
or βα → αα
or ( SQC ) ( DQC ) ( ZQC )
α1α 2 → β1β 2 α1β 2 → β1α 2
or β1β 2 → α 1α 2 or β1α 2 → α 1β 2
Wo
W1 W2 W1
zeroquantu m tr an siti ons
singlequantu m tr ansi tions
doublequantu m tr an siti ons
FIGURE 15. only zero. It can do so via zero.and doublequantum transitions lead to a change of the intensity of the spin1 transition (and hence to a NOE effect):
Seventh Chapter: The NOE Effect
Pg. single. Now.95
.
LECTURE COURSE: NMR SPECTROSCOPY
3. because relaxation of methyl protons is mainly governed by the other methyl protons. Optimize the lock power.5/ 5. If the NOE is very small.5/ 6.5/ 5.
1. that gives an intense lock signal (DMSO. Especially.
• • • • •
•
•
•
Seventh Chapter: The NOE Effect
Pg. the temperature must be stable. the mixing time has to be optimized for the molecule size. oxygen is a biradical).5/ 5. going from a nonviscous solvent (acetone) to a viscous solvent (DMSO) or measuring at lower temperatures may increase the size of the NOE dramatically (note that at low temperatures the danger is high that the temperature is not stable).5/ 6.5/ 6.5
2. irradiate the methyl group.5/ 2.7 Practical tips for NOE measurements: • • the conditions for measurement must be very stable (as always true for methods that rely on differences). D2O also has a large temperature shift of the solvent line. adjust lock power just below saturation to give a strong lock signal. rather not CDCl3 or D2O if possible). Pay attention to the choice of the solvent. Use sufficiently long relaxation delays (35 times T1). so that the lines easily shift when the temperature is not stable. Populations after zeroquantum (left). do not use too long mixing times in order to avoid spin diffusion.5
3. that means if the tumbling time is such that the NOE is near to the zerocrossing.5
6. acetone. For observation of NOE's between methyl groups and other protons. The sample should be concentrated enough but not too concentrated (little lock signal). For the same reason.5/ 3.5
5.5
3. never use spinning for NOE measurements! Measuring over night or on weekends is preferred because of less traffic in the building. Use a solvent.5
3/ 3
3/ 6
2.5
FIGURE 16. Avoid paramagnetic impurities! If very small effects should be measured.96
. singlequantum (middle) and doublequantum (right)
transitions and subsequent restoring due to spin2 irradiation.5
3/ 6
6/ 6
2. remove oxygen (degas the sample. because than the lock is more stable.
Newtons equation of motion is integrated:
mi
d r ( t ) = Fi ( t ) dt2 i
2
i = 1. Backbone atoms are calibrated with a r6 dependence.g. The force itself is expressed as the negative gradient of the potential energy. The volume of the NOESY crosspeaks may be expressed as
V = 〈 r 〉 f ( τc )
The distance between the two protons may vary due to ﬂexibility of the polypeptide chain. nowadays restrained molecular dynamics calculations are performed. The potential energy function is the normal force ﬁeld expanded by an additional term reﬂecting the nmr constraints:
U pot = U forcefield + U NMR
During a structure calculation a number of (typ. After the calculation the 2050 separately energyminimized conformers is superimposed for best ﬁt. The constant k can be determined on the basis of known distances (e. The inherently different ﬂexibility of different classes of protons in peptides (e. The next step is to convert NOESY peak volumes into distances. To calculate structures from these upper limits and torsion angle constraints distance geometry algorithms have been used in the past. the distance of sequential amide protons in helices is about 3Å). A resulting narrow structure bundle indicates good convergence of the calculation. a process known as “calibration”:
–6
V = k⁄d
4–6
These distances will then serve as upper limits to constrain distances between different protons in the calculated structures. N
Thereby. This is mostly the case when enough NOE’s are present to deﬁne the structure. that the strength of the NOE between protons depends on their distance. The NMR constrains in the potential energy function will force the molecule to adopt a structure that is compatible with the NMR data. Flexible parts of the molecule usually will display less NOE’s are less well deﬁned in the resulting structure bundle. 2050) randomly chosen conformers is energy minimized under a simulated annealing protocol. Due to the ﬂexible nature of polypeptides no lower limits are used (except for disulﬁde bridges).LECTURE COURSE: NMR SPECTROSCOPY
Excurs I: The use of NOE data for structure calculation of peptides and proteins
NMR data can be used for structure calculations of proteins up to about 30 kDa presently.g. Peaks are then integrated. whereas for NOE’s involving methyl protons a r4 calibration will be applied.
Seventh Chapter: The NOE Effect
Pg. Additionally. …. the coordinate of a atom at time t can be calculated from the force acting on it. The method uses the fact. scalar vicinal coupling constants may be used to constrain torsional angles. compare backbone atoms with methylgroups) is taken into account by applying different calibration functions for them. The ﬁrst step in the procedure is to assign the NOESY spectrum completely. In these calculations. The NOE depends also on the correlation time.97
. However.
parts of molecules that are far apart may be oriented relative to each other.98
. Bax et al. these dipolar interactions are averaged to zero in solution due to rapid tumbling and therefore rapid changes in the angle f. Relatively newly. Thereby. residual dipolar couplings have been used for describe orientations of parts of the molecule that are separated by a large distance.
2
–3
Seventh Chapter: The NOE Effect
Pg. only a few NOE’s between the two elements may be used. that it is possible to partially orient macromolecules in solutions containing bicelles (diskshaped vesicles). a reference frame for all protons (the alignment frame) is available and orientations are possible with respect to that frame irrespective of the number of intervening bonds. Marcomolecules in these solution are then partially oriented by simple steric reasons:
B0
φ
χ
The dipolar coupling can be calculated as:
A
δ dip ∝ S ( B 0 )γ A γ B r AB • [ χ A ( 3 cos 2 ( φ – 1 ) ) + χ B ( sin 2 φ cos 2φ ) ]
where cA and cB are the axial and rhombic alignment tensors and S the order parameter. the relative orientation of two helices) is difﬁcult to describe from NMR experiments. Dipolar interactions between two protons depend on the orientation of the vecto connecting the two atoms with respect to the external ﬁeld
Bo 1H
r
Bloc
=
φ
15N
µ (3 cos2 φ − 1) r3
Usually. Usually. However. Thereby.g.LECTURE COURSE: NMR SPECTROSCOPY
Excurs II: Use of dipolar couplings to yield structural constraints:
Orientations of structural elements with respect to each other (e. Any misassignment will lead to a large errors in the relative orientation. could show. These pospholipid vesicles orient in the magnetic ﬁeld such that the membrane normal is perpendicular to the direction of the magnetic ﬁeld.
sharp line positioned somewhere between the two lines) Intermediate exchange (a single. CHEMICAL OR CONFORMATIONAL EXCHANGE:
Let us assume that ethanol binds to a target molecule and dissociates of again:
OH CH 2 CH 3
+
CH3CH2OH
Me=1 ppm
Me=0. Three different states can be distinguished: • • Slow exchange (two sets of resonances with reasonable sharp lines) Fast exchange (a single. such as exchange between the hydroxyl protons and protons from dissolved water. Binding of methanol to protein in the vicinity of a aromatic residue leads to strong shift of methyl signal
In such a situation the methyl group of ethanol has different chemical environments in the bound and in the free state.5 ppm
FIGURE 1. In the bound state it is under the inﬂuence of the ring current shift of a tyrosine residue for example. and therefore the chemical shift will be lowﬁeld shifted. 1.99
.)
•
The underlying exchange process may also be chemical exchange. All these processes are frequently encountered in NMR. Alternatively it could be conformational exchange. very broad (or invisible) line.LECTURE COURSE: NMR SPECTROSCOPY
1. Depending on the exchange kinetics the methyl signal will appear as a single or as two resonances with more or less exchange broadening. In the free state the anisotropy effect due to the ring current shift has disappeared.1 Twosite exchange: Let as assume the following reaction:
+1 A → B −1 B → A
k
k
The lifetime of state A is:
τA
and of state B is
= 1 / k+1 = 1 / k−1
τB
Fifth Chapter: Chemical and conformational exchange
Pg.
The ratio is inﬂuenced by
•
increasing temperature accelerates the exchange reaction and therefore pushes the system into the fast exchange regime increasing static fields increases the line separation and therefore drives the system into the slow exchange regime. 2nd row from top: coalescence. Therein.100
Temp
.LECTURE COURSE: NMR SPECTROSCOPY
The NMR parameters that characterize both states are a) the chemical shift.
Fifth Chapter: Chemical and conformational exchange
Pg. Bottom: Slow exchange.spin couplings and c) the linewidth (transverse relaxation time). b) scalar spin.JB k >> 1/T2A1/T2B
The exchange time scale is deﬁned by the separation of the two signals relative to the exchange rate.JB k << 1/T2A1/T2B
k = δA . Top: Fast exchange.δB k= JA. The NMR timescale is deﬁned by the relation of the exchange rate with respect to the magnitude of the accompanying change in the corresponding NMR parameter: Exchange rates
Time scale slow intermediate fast
Chemical Shift Scalar Coupling Transverse Relax.
k << δA .δB k >> JA. a signal in the fast exchange regime is into the slow exchange regime pushed by decreasing the temperature or increasing the ﬁeld strength.JB k = 1/T2A1/T2B
k >> δA . At the coalescence temperature a very broad line with a plateau is observed. Appearance of twosite exchange spectra in dependence on the exchange rate.
•
The effect of chemical or conformational exchange on the chemical shift is shown below.δB k << JA.
fast exchange coalescence
Bo
slow exchange
FIGURE 2.
Through chemical exchange that proton is substituted by a proton that comes from water and is in the βstate.101
.1. Such an increase in reaction rate can be accomplished via a temperature increase and a decrease in frequency separation by lowering the magnitude of the static ﬁeld. In the ﬁgure below. This mechanism has been termed selfdecoupling and is the reason why OH protons in alcohols usually appear as (broad) singlets:
H CD3C D O H + H O H
H CD3C D
FIGURE 3. When the exchange is slow both lines of the doublet are visible but for the fast case only the average singlet will be observed. The exchange reaction causes an additional contribution to the linewidth of ∆ν 1 / 2 = 1 π (ν A − ν B ) 2 k+1
2
Thereby.LECTURE COURSE: NMR SPECTROSCOPY
1. only the avaraged line at the center without splitting due to coupling is visible. A similar situation exist with averaging of couplings:
J ave = p A J A + (1 − p A ) J B
An important example for averaging of couplings is found for hydroxyl protons of alcohols that exchange with solvent water protons.
O H
+
H
O H
Fifth Chapter: Chemical and conformational exchange
Pg. fast exchange regime) the narrower the line. the faster the reaction or the smaller the frequency difference (provided that a single line is observed. Exchange of the hydroxyl proton with a proton from water may lead to a change in the spin state of the OH proton.1 Fast exchange: At fast exchange a single resonance line is observed at the frequency
δ ave
= pAδ A + (1 − pA )δ B
It is obvious from the equation above that the resonance position of the averaged signal may not be simply the arithmetic mean of the resonance positions of the two slowexchanging lines because the population of the two states has to be taken into account (usually a unknown). the boxed proton feels the vicinal proton to be in the αstate (left side). If this process is fast.
A detailed investigation shows that it may be described as:
g(ν ) = 2τ A (ν A − ν X )
2 2 ν − 1 ν + ν + π 2τ 2 ν − ν ( A X ) A A 2
(
) (ν
2
−νX
)
2
The following ﬁgure shows calculated lineshapes for two signals 10 Hz apart (top trace) depending on the lifetime (which is the inverse of the reaction rate) or for two signals with 0. 10 Hz separated appear as a singlet.1sec lifetime depending on the frequency separation (given in Hz):
0.1. At slightly acidic pH the amide protons give separate. Bottom: Calculated for a lifetime of 0.1 ∆δ =10 Hz
0.
At coalescence the lineshape is characterized by a ﬂat top. You
Fifth Chapter: Chemical and conformational exchange
Pg. one is not interested in recording spectra with exchangebroadened lines.5
1. A typical example where both situations (slow and fast exchange) are simultaneously encountered is exchange of amide and hydroxyl protons of peptides with solvent in aqueous solution. The corresponding lifetime at coalescence is given by
τc A
= 2 (ν − ν X ) π A
Usually.01
0. 1. Top: Two lines. sharp lines and the exchange rate is on the order of 110 sec1. if the lifetime of the separate states is short (0.1 sec
FIGURE 4. temperature decrease or ﬁeld increase will lead to a sharpening of the lines.085
0. exchange between hydroxyl and water protons is so fast that the hydroxyl protons coincide with the water chemical shift and cannot be observed separately.102
.05
0.3 The intermediate case: The lineshape of the signals changes upon passing from the slow into the fast exchange regime.3
0.1 s with varying frequency separations (115 Hz).LECTURE COURSE: NMR SPECTROSCOPY
1.2
τa
0.01s) or as two separate lines.0
∆δ
1
3
4
5
6
10
15
τ a=0.1. when the lifetime is long (1s).2 The slow exchange limit: When two separate signals are observed the additional contribution to the linewidth due to exchange is: ∆ν = k+1 π
Hence. In contrast.
since the exchange rate is normally much faster than the crossrelaxation rate. record spectra at lower field. the mixing time is usually chosen shorter. When that temperature cannot be reached (sample not heatstable or solvent is volatile). Crosspeaks are due to exchanging (chemical
Fifth Chapter: Chemical and conformational exchange
Pg.295K
600MHz. The following ﬁgure shows 1D spectra (expansions) of chinin recorded at 300 and 600 MHz.103
. when the lines get broader upon temperature rise cool down or proceed at higher field.2. However.
•
If you cannot get spectra with reasonable sharp lines following this procedure it is worth trying to change the solvent which may have a dramatic effect.300K
3.2 Investigation of exchange processes: 1.5
2.
300MHz.5
3.320K
600MHz.LECTURE COURSE: NMR SPECTROSCOPY
can ﬁnd out the proper measuring conditions by • recording spectra at higher temperature ==>when the lines get sharper heat until the lines are sufficiently narrow.0
ppm
FIGURE 5. Appearance of spectra displaying exchange phenomena in dependence on the static field
1. It is obvious that by going from 600 to 300 MHz a considerable improvement in linewidths has been achieved.0
2. Often cyclic compounds display such dynamic behavior due to slow conformational interconversions.310K
300MHz.1 EXSY spectroscopy: The 2D EXSY experiment is in principle identical to the NOESY pulse experiment.
Singleline spectra of resonance A (left) and B (right)after various time increments. right:Expansion
1. The middle trace displays a dramatic effect on resonance A when exchange occurs. The top trace shows the case without exchange between A and B (no effect on A).5
7
6
5
4
3
2 ppm
7.0
7. Middle:Exchange.
ppm 2 4 6
ppm
7. However.T1=inf. The signal is shown after successive time increments for resonance A (left) or resonance B (right). the longitudinal relaxFifth Chapter: Chemical and conformational exchange Pg.5
7. by application of a selective 180 deg. pulse). it recovers with T1.g.2 Saturation transfer: When a signal is inverted (e. bottom: exchange.2.
kex =0. However. T1=1s. other protons that are in exchange with the inverted or saturated resonance become also saturated or inverted:
A
B
kex=0
A
B
kex=0.
In this ﬁgure resonance B has been inverted. 2D EXSY:left: Spectrum overview.104
.0
ppm
FIGURE 6. Top: No exchange. infinitely long T1.LECTURE COURSE: NMR SPECTROSCOPY
or conformational) protons.T1=1s
FIGURE 7.
1.g.76 + ∆ S#/R). In the lower trace the signal A recovers due to T1 relaxation.3 Determination of activation parameters: The observation of dynamic processes in NMR spectra allows to calculate parameters for the activation parameters of the exchange process.96 + ln [ Jmol ] νA −νB
Fifth Chapter: Chemical and conformational exchange
Pg. Hydroxyl protons that exchange with the solvent (e. A frequent case where saturation transfer occurs is through presaturation of solvent.
= κ
kT − ∆H # / RT ∆S # / R e e h
κ is the transmission coefficient (usually 1) k the Boltzmann constant T the temperature h the Planck constant A plot of ln(k/T) versus 1/T yields a line with slope (. In close agreement the activation energy can be estimated as
∆G #
≈
Tc −1 RTc 22. This is related to the reaction rate k via
k
= κ
kT − ∆G # / RT e h
Theory of thermodynamics states that
∆G #
so that
= ∆H # − T∆S #
k
therein. The activation energy for such a process can be calculated according to the Arhenius equation
Ea = − RT ln K #
in which K# is the equilibrium constant for the formation of the activated complex. water) vanish from the spectrum or largely decrease in intensity when solvent presaturation is applied. From the rate of recovery the exchange rate can be calculated (provided T1 has been determined before).105
.LECTURE COURSE: NMR SPECTROSCOPY
ation time T1 has been assumed to be inﬁnity.∆ H#/R) and intercept (23.
relaxation effects are usually ignored but can be added easily. THE PRODUCT OPERATOR FORMALISM (POF) FOR DESCRIPTION OF PULSEEXPERIMENTS:
Simple onedimensional NMR experiments may be conveniently described using the pictorial vector description. In the POF. precession and scalar coupling can be calculated in arbitrary order.106
.LECTURE COURSE: NMR SPECTROSCOPY
1. which is the ensemble property formed by adding up all dipole moments from separate spin. The following events may happen during or after application of radiofrequency (RF) pulses: • • • • chemical shift precession evolution of scalar or dipolar couplings change of the state of the spins due to additional RF fields relaxation
In the following we will see how these events are described in the product operator formalism (POF). The transformations due to shift precession or coupling are described as rotations in threedimensional cartesian space:
x
x
∆t=1/4ν y y
FIGURE 1. the POF may only be used to describe spin systems in the weak coupling case (no second order effects). If not necessary.and cosine modulated function. However. Truly speaking. in order to more deeply understand twodimensional NMR or multiplequantum spectroscopy the product operator formalism is required. rather than on individual spins. Rotation of a vector after a time 1/4ν
We have seen before that a continous rotation can be described as an harmonic oscillation and presents a superpostion of a sine.
Eighth Chapter: The product operator formalism Pg. In the POF calculations are done on magnetization.
2 Chemical shift precession: After application of a 90˚ RF pulse spins are correlated in phase leading to a bunching of phases. 1.
γB1 2π
=
1 PW (360°)
The sign convention for rotations is that applying a ﬁeld along the xaxis will rotate in clockwise sense (Field along y: Look down the yaxis and rotate clockwise. from z to x) 1.1 RF pulses: Application of a RF ﬁeld along the xaxis will rotate zmagnetization in the z.y plane:
Bo=z Bo= z
Bo= z
x y
x y
x y
FIGURE 2.107
. The resulting magnetization vector rotates in the x. The ﬁeld strength is expressed in terms of the frequency. The frequency of rotation depends on the strength of the ﬁeld. which is the inverse of the time required for a 360˚ rotation. e. This will achieve the following conversions: z > y > z > y > z
It is described as:
Iz
βI x →
Iz cos β
−
I y sin β
Of particular importance are rotations of 90 and 180 degrees. Rotation of zmagnetization about the xaxis.LECTURE COURSE: NMR SPECTROSCOPY
In the example given above a property aligned along the xaxis rotates about the zaxis onto the yaxis with a frequency ω.g. In the POF this will read as follows: Ax
A ω→ z
Ax cos(ωt ) +
Ay sin(ωt )
The symbol over the arrow indicates the axis and the frequency of rotation.y plane
Eighth Chapter: The product operator formalism Pg.
g. INEPT. Left: Equilibrium state.108
.3 Scalar spin. 1. After a certain amount of time it will have rotated by a speciﬁc angle depending on its offset frequency. DEPT) or the mixing
Eighth Chapter: The product operator formalism Pg. Note that chemical shift evolution can only take place for transverse magnetization!
Bo =z
B o= z
x y
x y
x
y
FIGURE 3.LECTURE COURSE: NMR SPECTROSCOPY
(transverse plane) with a certain angular velocity. Having different energies means that these two different kinds of spins precess with different frequencies because frequency and energy are related to each other. Middle: State after 90 deg RF pulse. Right: Shift precession in the
transverse plane.
Shift precession amounts to a rotation of the magnetization vector in the transverse plane. When both vectors are 180˚ out of phase the resulting term is I y Sα + ( − I y )S β = 2 I y ( Sα − S β ) = 2 I y Sz
This state is of particular importance for coherence transfer which is the crucial step for polarization transfer experiments (e. Ix ω I z → I x cos(ωt ) + I y sin(ωt )
The precession frequency is ω=2πδ where δ is the offset from the transmitter frequency.spin coupling: Due to the fermicontact interaction spins that have a neigbouring spin in the αstate are of slightly different energy than those that have them in the βstate. In a reference coordinate system that rotates with the center frequency of a dublet the downﬁeld component will rotate with a frequency of +J/2 in one direction and the other component with –J/2 in the other direction.
LECTURE COURSE: NMR SPECTROSCOPY
steps in 2D experiments.Xt) leads to the following signal: JI.r. Scalar couplings do occur as inphase splitting when the operator is Ixcos(πJI. to S) 2IzSzlongitudinal twospin order 2IxSytwospin coherence (mixture of zeroand doublequantum coherence.X JI. Iytransverse x (or y) magnetization Izlongitudinal magnetization 2IxSzantiphase I magnetization (or I magnetization antiphase w.S JI. Vectorial representation of the states due to evolution of scalar couplings
A number of operators are frequently encountered during the calculations: • • • • • • Ix.109
. multiplequantum coherence)
Two operators that are both transverse cannot evolve scalar couplings amongst each other! The product operators can be directly related to the appearance of signals.spin coupling is described as:
JI πz Sz → I x cos(πJt ) + 2 I y Sz sin(πJt ) JI πz Sz → − I x
Ix
( I( Sα)
x
x y Ix
y
Σ=
x y I(Sβ) y 2IySz
x
x y I x
FIGURE 4. In the product operator formalism the transformation under scalar spin.S JI.S
Eighth Chapter: The product operator formalism
Pg.St)cos(πJI.St) or as antiphase splitting if the operator is 2IySzsin( πJI.St) The operator 2IySzsin(πJI.
LECTURE COURSE: NMR SPECTROSCOPY
1. A 90 degree RF pulse applied along the xaxis is immediately followed by data acquisition. However. shaded rectangles. The events are subsequently calculated: • 90 degree xpulse: Iz • chemical shift evolution − Iy • ωtI z → − I y cos(ωt ) + I x sin(ωt )
I 90(→ − I y x )
scalar spin. Using the POF we can summarize the events for a 2spin system. Sy.spin coupling:
I x sin(ωt )
Jt ( I z S z π)→ − I y cos(ωt )cos(πJt ) + 2 I x Sz cos(ωt )sin(πJt )
− I y cos(ωt ) +
+ I x sin(ωt )cos(πJt ) + 2 I y Sz sin(ωt )sin(πJt )
Note that chemical shift and scalar coupling may be calculated in arbitrary order (but not the effect of pulses and shift/coupling!). During a pulse sequence all possible interactions have to be calculated which may lead to a huge number of terms.4 A simple onedimensional NMR experiment: A conventional onedimensional experiment for recording proton spectra looks like the following:
x
FIGURE 5. 90 degree pulses are drawn as narrow. only a limited number of terms lead to detectable magnetization at the end and only those are of interest because they contribute to the signal: • • • directly observable are only single operators that contain transverse terms (Ix. Usually. one starts with pure zmagnetization. wider rectangles)..Iy. Sx. pulses as black.110
Eighth Chapter: The product operator formalism
.
(In the following. 180 deg. A simple 1D experiment consisting of a 90° pulse followed by sampling of the FID.) antiphase magnetization (such as IySz) evolves into inphase magnetization (Ix ) if the corresponding coupling is resolved terms that contain more than one transverse operator are not observable and hence can be ignored in the end of the sequence
Pg.
5 The effect of 180 degree pulses: 180 degree pulses are often used to • • refocus chemical shift refocus scalar couplings
When a 180 degree pulse is placed in the centre of a delay it will refocus chemical shift evolution of transverse magnetization (see left part of the following ﬁgure). Only the latter leads to decoupling. In this context it is very important to distinguish two cases: • both spins I and S experience the 180 degree pulse which is usually the case if they are both protons (homonuclear case) only one of the two nuclei experiences the pulse which is the case for a 1H.
Eighth Chapter: The product operator formalism Pg.into the βstate: Iα 180→ I β Iβ 180→ I α
The effect of 180 degree pulses are summarized in the following ﬁgure:
I
∆
∆
∆
∆
∆
∆
S
refocussing of I shift refocussing of I.spin coupling are due to different spins that have neighbouring spins in either the α.S coupling refocussing of I shift evolution of I.13C pair. 180 degree pulses will ﬂip spins from the α.S coupling I shift evolution refocussing of I.or the βstate. In the ﬁrst case both spins are simultaneously ﬂipped and no overall effect occurs.into the βstate or vice versa the coupling evolution will be refocussed after a time that is the same as the one during which the system was allowed to evolve the coupling. When this coupling partner is ﬂipped from the α.LECTURE COURSE: NMR SPECTROSCOPY
Certain terms that are of negligible intensity can be removed during the calculation thereby largely reducing the number of operators.S coupling
FIGURE 6. 1. Effects of 180° pulse(s) on shift precession and evolution of scalar couplings
We have seen before that the two dublet lines arising from spin.111
•
.
112
.
Eighth Chapter: The product operator formalism
Pg. Otherwise. S )→ 2( − I y )( − Sz ) ∆ → − I x
1. multiplequantum coherences are formed: 2 I y Sz 2 I y Sz
y → 2 I y Sz
90° ( I )
[2 b ] [3b]
90°( S x )→ − 2 I y Sy
Through the coherence transfer step transverse Ispin magnetization has been transformed into transverse Sspin magnetization. Note that in the homonuclear case steps [2] and [3] can be achieved with a single pulse. Antiphase Ispin coherence is then transferred into antiphase Sspin coherence:
Ix
πJt ) I z S (z →
I x cos(πJt ) + 2 I y Sz sin(πJt )
[1]
2 I y Sz 2 Iz Sz
90°( I x )→ 2 Iz Sz 90°( S x )→ − 2 Iz Sy
[2 a ] [3a]
When the time t during which scalar coupling takes place is exactly equal to (2J)1 sin(πJt)=sin(π/2) and Ispin coherence has quantitatively been transformed into antiphase coherence. homonuclear scalar couplings are not refocussed since both spins are ﬂipped: Ix ∆ → 2 I y Sz 180( I .LECTURE COURSE: NMR SPECTROSCOPY
The refocussing of chemical shift can be described with the product operator formalism easily in the following way: Ix ∆ → I y 180( x )→ − I y ∆ → I x
and for evolution of heteronuclear scalar couplings: Ix ∆ → 2 I y Sz 180( S )→ 2 I y ( − Sz ) ∆ → I x
However.DEPT) multidimensional correlation experiments
In order to transfer coherence couplings must have been evolved.6 Coherence transfer: Coherence transfer is the crucial step of • • polarization transfer experiments (INEPT. It is also important to realize that step [2] requires the phase of the 90˚ pulse to be x.
13C
spectra are much lower in sensitivity because • • the natural abundance of 13C is 1/100 of that of protons the energy separation between α.
Polarization transfer is aimed at bringing the population difference from protons to carbons and hence increasing the sensitivity.7 Polarization transfer: Polarization transfer is a common building block in pulseexperiments to increase sensitivity. We have seen before that this number depends on the relative populations of α.113
. The higher the energy gap between the two states the larger the population difference and hence the stronger the intensity of the signal.LECTURE COURSE: NMR SPECTROSCOPY
1. The sensitivity is proportional to the number of spins that can undergo a certain transition. we need a 180˚ pulse in the center of the proton delay.and βstates according to the Boltzmann distribution. However.S )→ 2 I x Sz 1
y → − 2 Iz Sz
90° ( I )
90°( S x )→ 2 Iz Sy
In principle. this
Eighth Chapter: The product operator formalism Pg. However.and βstates is γ(13C)/γ(1H)=1/4 and hence the number of transitions are much lower.13C polarization transfer).H) 1/4 J(C. one might be tempted to remove the 180˚ pulses on the proton and carbon channel. S )
− 2 I y Sy sin(ωt )
Therefore. The prototype for the PT sequences is the INEPT experiment:
x y 1/4 J(C. Pulse sequence for INEPT experiment (1H.
Using the POF the INEPT sequence can be summarized in the following way: Iz 90°( I x )→ − I y
2 J ( I.H)
1H
13C
FIGURE 7. chemical shift evolution would then take place for protons transforming 2 I x Sz ωtI z → 2 I x Sz cos(ωt ) + 2 I y Sz sin(ωt )
The second term would be transformed into nonobservable multiplequantum coherence through the cascade of the following two 90˚ pulses:
2 I y Sz sin(ωt )
y x →
90° ( I .
The important achievement of the PT sequence is that the amount of antiphase carbon magnetization is proportional to the starting Ispin magnetization which is Iz=(IαIβ). Hence.CH2 and CH carbons on the proton pulse length is shown below:
CH
CH3
CH2 45° 90° 135°
FIGURE 8. For β=135 degrees. For β =90 degrees. a large variation of coupling constants leads to crosstalk in the edited spectra. The DEPT experiment allows to record
13C
spectra that are edited
with respect to the number of protons that are directly bonded to the carbons. and we do need the coupling evolution for the transfer. At the end of the PT sequence the amount of transverse cabon magnetization depends on the Boltzmann distribution of the proton levels which is much more favourable! An experiment that has become even more popular than INEPT is the DEPT sequence. The DEPT sequence relies on uniform 1J (CH) coupling constants.LECTURE COURSE: NMR SPECTROSCOPY
proton pulse would unfortunately decouple protons from carbons. a DEPT135 experiment is mostly sufﬁcient but should be complemented by a DEPT90 in case of ambiguities for CH3/CH. methyl and methin carbons are positive and methylen carbons are negative. Transfer amplitudes for CHn (n=13) groups in DEPT experiments in dependency of the flip angle of the final proton pulse.
Since CH and CH3 carbons can usually be distinguished on the base of their chemical shifts. a double PT 2D experiment can be used to increase the senitivity
Eighth Chapter: The product operator formalism
Pg. Nonproton bearing carbons are missing in DEPT spectra. The dependence of the signal intensity for CH3. only CH carbons are contained. Of course. The editing is achieved through suitable choice of the length of the ﬁnal proton pulse in the sequence.114
. a 180˚ pulse on the carbon channel is additionally required in order to avoid decoupling.
LECTURE COURSE: NMR SPECTROSCOPY
further such as the HSQC experiment:
Sensitivity (fully relaxed. and with PT (INEPT) and with 1H detection (2*PT. Comparison of sensitivity of carbondetection experiments without PT ( inversegated 13C.115
HSQC
13C
1H
1H
t1
Decoupling
. HSQC)
Eighth Chapter: The product operator formalism
INEPT
Pg. 13C{1H}). 100% isotopic abundance) γ(13C)5/2
γ(13C)5/2 + NOE
γ(1H)(13C)3/2
γ(1H)5/2
RD
Decoupling
Decoupling
Decoupling
RD
RD
inversegated 13C
1H
13C
13C
1H
RD
13C
13C{1 H}
FIGURE 9.
Assume we have two state vectors. <ψ'ψ’’> is interpreted as the probability to go from state ψ’ to ψ’’. one describing the inital state of the spin system. The scalar product of two different states is 〈ψ'ψ''〉 = 〈ψ'ψ'〉 + 〈ψ''ψ''〉 + 2 〈ψ'ψ'〉
1⁄2 1⁄2
〈ψ''ψ''〉
cos φ
in which the component printed in bold indicates the amount of interference between the two states. ψ> is called a state vector and α> and β> are the basis states. The scalar product of the two vectors is deﬁned as: 〈ψ  ψ 〉
2 2 2
= c1 + c2
which is the square of the length (norm) of the state vector ψ: α>
C1
α> ψ> “ ψ> ψtot>
φ
C2
‘ ψ> β>
β>
The length of the state vector ψ> is interpreted as the probability of ﬁnding the spin system in a particular state.LECTURE COURSE: NMR SPECTROSCOPY
Excurs: The foundations of the product operator formalism: The wavefunction for the state of a onespin system can be formulated as ψ 〉 = c 1 α 〉 + c 2 β 〉 in which the coefﬁcients c1 and c2 indicate to which extent the spin occupies the α or β state. The basis states are orthogonal vectors in Hilbert space (a Ndimensional vector space for a system with N basis states with some special properties). We can now deﬁne an operator A that transforms ψ'' into ψ’: ψ''〉 = A ψ'〉 Often in NMR these transformations are rotations in 3D space that do not change the length of the vectors (socalled “unitary transformations”) and the operator is a rotation matrix. one describing the ﬁnal state: ψ i〉 = c 1i α〉 + c 2i β〉 ψ f〉 = c 1f α〉 + c 2f β〉
Eighth Chapter: The product operator formalism
Pg.116
.
). shift etc. Cp = 0 or Cq p → Cq cosθ
C C
[
]
+ i Cq . Cp sin θ
[
]
if Cq . σ ( t 0 ) ] = – i ( Hσ – σH ) The time evolution of the spin states can be expressed as:
Cq p → Cq if Cq .117
. Cp ≠ 0
[
]
The basic rotations are: z 2IzSz z
x
y
Ix
2IySz
Eighth Chapter: The product operator formalism
Pg.LECTURE COURSE: NMR SPECTROSCOPY
To mathematically express the transformation we can arrange the state vector of the initial state as a column vector and the state vector ofthe ﬁnal state in form of a row vector and write: c 1i c 2i A 11 A 12 A 21 A 22 = c 1f c 2f
The timeevolution of the density matrix is described by the Liouvillevan Neumann Equation: d σ ( t ) = – i [ H. σ ( t 0 ) ] dt in which H is the Hamiltonian describing the interaction (coupling. The commutator – i [ H.
118
. TWODIMENSIONAL NMR SPECTROSCOPY:
The introduction of twodimensional NMR spectroscopy has largely increased the potential power of NMR as a tool for structure elucidation of larger molecules. which allows to correlate frequencies:
ΩΑ
ΩΒ
ΩΒ
C
D
F1
D C
ΩΑ
F2
FIGURE 1. It's main advantages are: • • dispersion of signals into two orthogonal dimensions and identification of correlations
The 2dimensional NMR experiment is characterized by the introduction of a second frequency axis.
The two frequency domains are called the direct (F2) and the indirect (F1) frequency domains. Appearance of homonuclear COSY spectrum with crosspeaks (C) and diagonalpeaks(D). F1=ΩB are called crosspeaks. Homonuclear 2D spectra are usually symmetric about the diagonal. Frequencies of signals in the direct dimension have been directly detected in the receiver coil. The diagonal contains the onedimensional spectrum. Twodimensional experiments can be classiﬁed into certain groups:
Ninth Chapter: 2 dimensional NMR experiments
Pg. those of the indirect dimension were derived from the second Fourier transform of the amplitude modulated signals. and they indicate the spins with frequencies ΩA and ΩB are correlated. Offdiagonal peaks at the frequency F2=ΩA.LECTURE COURSE: NMR SPECTROSCOPY
1.
that separate chemical shift and Jcoupling information in two orthogonal frequency dimensions (F2: chemical shift of spin A.13C correlations) in different frequency dimensions.1H COSY. F1: chemical shift of spin B for crosspeaks) Jresolved experiments. The signal of the spin B. varies in amplitude from experiment to experiment. in which a delay called the evolution time is systematically incremented. F1: Jcoupling of spin A).
•
Similarly experiments can be subdivided into
•
shiftcorrelated 2D experiments. which is detected. then chemical shift labelled during the evolution period.119
. The 2D data matrix is recorded by performing a set of 1D spectra.LECTURE COURSE: NMR SPECTROSCOPY
• homonuclear correlated spectra that contain frequencies of similar nuclei in both dimensions (e. Building blocks of 2D experiment. magnetization is detected on the spin B.
•
Each pulsesequence for a 2D experiment contains the basic elements
Preparation
(Excitation of spin A)
Evolution
(Chemical Shift labelling of spin A)
(Coherence transfer to spin B)
Mixing
Detection
FIGURE 2.g. F2=ΩB crosspeak the spin A is excited in the preparation period.
For the example of the F1=ΩA. Finally. INADEQUATE) heteronuclear correlated spectra that contain frequencies of different nuclei (e.g. magnetization is transferred from spin A to spin B in the mixing period. that link two spins via their chemical shifts (F2: chemical shift of spin A. 1H. 1H.
Ninth Chapter: 2 dimensional NMR experiments Pg. Subsequently.
The 2D spectrum is yielded from a twodimensional Fourier transformation:
FIGURE 3.120
.proton correlation experiment like the COSY. it is a simple 90 degree pulse for excitation.1 The preparation period: In principle. In a proton. Some experiments that correlate carbon (or nitrogen) with proton frequencies use an initial INEPT polarization transfer from proton to carbon to increase sensitivity (dramatically). These experiments are the so
Ninth Chapter: 2 dimensional NMR experiments
Pg.0. the preparation period serves to create transverse coherences.LECTURE COURSE: NMR SPECTROSCOPY
and the modulation frequency of the signal intensity corresponds to the chemical shift of the spin A. Translation of the amplitude modulation of a signal into the frequency of the indirect
dimension through Fourier transform.
1.
In order to understand how frequency labelling in the indirect dimension is achieved.LECTURE COURSE: NMR SPECTROSCOPY
called inversedetection experiments. Fourier transform of the t1domain data yields the frequency dimension F1 and t2 corresponds to F2. Sampling of data in the direct dimension. S )→ 2 I x Sz ∆
y y → 2 Iz Sx
90° ( I . the width of the spectrum in Hz): dw = 1 SW
The resolution depends on the number of data points sampled and the spectral width (neglecting relaxation effects). In the product operator formalism the preparation is described as Iz 90° x → − I y
or in the case of inversedetection experiments:
Iz 90°( I x )→ − Iy
− ∆ −180 ( I . Usually.
Consecutive data points are separated by a dwell time.2 The evolution period: The evolution period is the pulse sequence element that enables frequency labelling in the indirect dimension. Frequency labelling in the indirect dimension is done analogously. The signal is sampled in discontinuous mode:
(td =16)
DW
AQ
FIGURE 4. However. it is useful to recall how the frequencies in the direct dimension are sampled. S )
1.121
. the corresponding time is called t1 in contrast to t2 in the direct detection dimension.0.
Ninth Chapter: 2 dimensional NMR experiments Pg. The length of the dwell time is related to the spectral width (SW.
the term just before the transfer step can also be IySz(instead of IxSz). transverse Ispin coherence is transferred into transverse Sspin coherence. In the product operator formalism frequency labelling is described as: Ix t1 → I x cos(ω I t1 ) + I y sin(ω I t1 )
1.LECTURE COURSE: NMR SPECTROSCOPY
data points are taken in separate 1D experiments. Application of a 90° pulse with phase y on the I and S spin will give IySx.122
.3 The mixing period: The many 2D experiments differ in the way they transfer magnetization from spin A to spin B. which cannot
Ninth Chapter: 2 dimensional NMR experiments Pg. Because the mixing step separates the t1 and t2 periods. However. Since the evolution time is incremented systematically in successive FIDs the frequency in the indirect dimension is sampled analogously to the one of the direct dimension. During a 2D experiment a series of 1D spectra is recorded which differ by the fact that the evolution time has been systematically incremented from experiment to experiment. Depending on the length of the evolution period more or less of the magnetization will be turned onto the zaxis thereby modulating the intensity of the remaining signal. Operators like IxSz evolve with the chemical shift of the spin I whereas operators like IzSx evolve with the shift of spin S. that means in the construction of the mixing process. The signal precesses during t1.0.and zeroquantum coherence. S )
Herein. a mixture of double. Possible transfersequences may rely on • • • scalar couplings (COSY. such a transfer step will lead to signals corresponding to crosspeaks after 2D FT. This is schematically shown in the ﬁgure on the following page for signals that lead to diagonal peaks (no magnetization transfer during the sequence). depending on the chemical shift evolution of the I spin in t1. TOCSYtype experiments) dipolar couplings (NOESY/ROESY type experiments) chemical exchanges (EXSY type experiments)
In product operator language the mixing for the COSYtype experiments is described as 2 I x Sz
y y → 2 Iz Sx
90° ( I .
FID in F1
FT(F2)
1
2
3
4
5
FIGURE 5.LECTURE COURSE: NMR SPECTROSCOPY
be observed. COSYtype mixing in the vector model
Ninth Chapter: 2 dimensional NMR experiments
Pg.123
PREPARATION
EVOLUTION
MIXING
DETECTION
FID in F2
.
the sensitivity is increased according to Int ∝ γ ex
3/ γ det2
In addition.0.4 The detection period: The detection period simply comprises acquisition of the FID with or without heteronuclear decoupling. HMQC.5 Hetcor and inversedetection experiments: The heteronuclear correlation experiments (Hetcor.
1. Magnetization flow for coherence transfer between spins A and B. Inverse detection experiments include an additional INEPTtype protonheteronucleus polarization transfer step. However.124
. the mixing pulse must be applied for both kinds of nuclei (1H and 13C) separately.LECTURE COURSE: NMR SPECTROSCOPY
PREPARATION
EVOLUTION
MIXING
Spin A
α β β
α
α Spin B β
FIGURE 6.0.
1.
The TOCSY transfer is different in the way that inphase magnetization is transferred at twice the rate: Ix
180° " "→ Sx
For the NOESY mixing the description is Iz τ m → ( − )Sz
Some of the more advanced tripleresonance experiments mixing includes magnetization relay via many nuclei of possibly different nature. Thereby. etc.) are in principle of the COSYtype. faster pulsing is possible since the proton T1's are usually much
Ninth Chapter: 2 dimensional NMR experiments
Pg.
The prototype inverse detection experiment.1H] spectra is suppression of protons bound to
12
C.125
. the HSQC (heteronuclear singlequantum coherence) experiment is shown below.1 Phasecycling: A large problem in [13C.
Ninth Chapter: 2 dimensional NMR experiments
Pg. The experiment contains two polarization transfer (INEPT) steps. Building blocks of homonuclear (left) and heteronuclear (middle and right) experiments.
Preparation INEPT Evolution ReINEPT Detection
∆
∆
t1
∆
∆
DEC 1H 13C Hz Hy 2HxCz 2H zC y 2H yCz cos(ωC t1) 2H zC ycos(ωC t1) 2HyCz cos(ωC t1)cos(ω Ht2)
FIGURE 8.LECTURE COURSE: NMR SPECTROSCOPY
shorter than those of 13C or 15N. Building block of HSQC experiment.
homonuclear heteronuclear (X detection mode)
Preparation
heteronuclear (1 H detection mode)
Preparation
Excitation
Evolution
Evolution
PTTransfer to Xnucleus
Mixing
PTTransfer to Xnucleus
Evolution
Detection
Detection
PTTransfer back to proton
Detection
FIGURE 7.
1.
Clearly.LECTURE COURSE: NMR SPECTROSCOPY
This is achieved through phasecycling of the ﬁrst 90˚ pulse on the carbon channel. Usually the NMR spectroscopist spends a considerable amount of time to have maximum ﬁeld homogeneity over the sample volume. The satellite lines (those proton lines. unstable measuring conditions will cause incomplete cancellation.2 An Alternative: Pulsed Field Gradients By applying pulsed ﬁeld gradients the homogeneity of the external magnetic ﬁeld is destroyed and spins with the same chemical shift are defocused to a degree which depends on their location in the probe. The crucial point is that the amount of dephasing is proportional to the coherence level of the spins at the time the gradient is applied. When the receiver phase is inverted too addition of the two FIDs leads to cancellation of the central proton line (the one that belongs to the 1H12C fragment). Coherence selection in HSQC spectra and origin of t1 noise in HSQC spectra and
1. If such a gradient would be applied during signal acquisition a broad signal corresponding to a wide range of frequencies would be observed instead of a sharp line. and the centre signal will show up as t1 noise:
FID1
φ 1 =x φ Rec =x
1 J C. Doublequantum coherences are dephased by twice the amount than single
Ninth Chapter: 2 dimensional NMR experiments
Pg. which belong to the 1H13C fragment) are inverted when the phase of the ﬁrst 90˚ carbon pulse is shifted by 180˚. By detuning the zshim starting from a perfectly shimmed magnet a linear B0 gradient is created along the sample axis so that spins in the upper part of the tube have higher frequencies than those at the bottom.H φ 1 =x φ Rec =x
FID2
1 H 13 C
∆
∆
∆
∆
φ
t1 HSQC DEC
Σ
FIGURE 9.126
.
proton coherences can be labelled using a pulsed ﬁeld gradient. zeroquantum coherences are not dephased at all. carbon single quantum coherences can be refocused by a gradient of opposite sign and appropriate strength. In the doublequantum ﬁltered COSY experiment a gradient is applied during the time doublequantum coherence is exists. after magnetization transfer to carbon13. and. Usually at least two gradients are applied during the sequence. The 2:1 ratio of the two gradients ensures that the desired coherence pathway has been selected:
A
B
C
t1
D
Gz
BO
Gz
BO
FIGURE 10.
Ninth Chapter: 2 dimensional NMR experiments Pg. Alternatively. Because spectra are recorded without addition/subtraction of scans. it's coordinate Zi in the probe and the duration τ: ∆υ ∝ pi γ i Zi Gzτ By applying a gradient of the same strength but opposite sign. the gyromagnetic ratio γi of the spin. Since dephasing and rephasing is done within the same scan.127
. no further scans are needed provided the S/N is sufﬁcient.LECTURE COURSE: NMR SPECTROSCOPY
quantum coherences. coherences of the same order are rephased again. At the end of the sequence (when singlequantum coherences are existent) a readout gradient is used of half the strength to refocus the gradient.
The change of the lamor frequency is proportional to the order of coherence pi. Effect of ﬁeld gradients on isochronous spins at different locations in the probe. the strength and sign of the applied linear ﬁeld gradient pulse Gz. (D) vector representation of isochronous spins during the experiment. (A)
DQFCOSY sequence with application of gradients (B). (C) selected coherence transfer pathway.
This is especially helpful in case of overlap of proton frequencies.g.1H] HSQCTOCSY experiment correlates not only the anomeric proton with its carbon but also with the neighboring protons. Then. In case of isotropic enrichment. For example. not only the direct (1J(C. 1.3 Hybrid 2D techniques: Hybrid experiments are very similar to threedimensional NMR experiments. 12ms) coherence transfer in HSQCTOCSY experiments is limited to neighboring protons. in which the second time variable is kept constant.128
. as is the case for NOESY or TOCSY experiments. Obviously. Classical examples are HMQC/HSQC experiments coupled with a subsequent TOCSY or NOESY (ROESY) transfer step. a HSQCNOESY on natural abundance is very insensitive. information from such an experiment is similar to that of COSY spectra but signals are dispersed along the carbon dimension making it a very valuable experiments for (oligo)saccharides or peptides.LECTURE COURSE: NMR SPECTROSCOPY
instrumental instabilities are less pronounced and therefore twodimensional experiments tend to contain much less t1 noise. but also connectivies to other (mostly vicinal) protons of the proton spin system. Furthermore. A second disadvantage stems from the relatively long duration of gradients and the time required for reestablishing the homogenous ﬁeld. no saturation transfer due to fast exchanging protons is possible and signals with identical chemical shifts to those of the solvent can easily be observed. One obviously useful ﬁeld for application lies in oligosaccharide NMR. the sensitivity of the correlations is reduced when compared with the HSQC experiment. Whenever dephasing and rephasing intervals are separated by a long delay.H) connectivities are observed.
Ninth Chapter: 2 dimensional NMR experiments
Pg. a [13C. diffusion leads to a signiﬁcant loss of magnetization. In these experiment.When the mixing time is limited (e. such a HSQCNOESY can be a rather sensitive experiment for larger molecules (those that give strong NOE enhancements). solvent suppression is superior. For small molecules with small NOE enhancements.
for long mixing times all protons within the same spinsystem are correlated doublequantum spectroscopy identiﬁcation of degenerate methylene protons TQFCOSY identiﬁcation of glycine residues in peptides Xﬁltered Exp.129
F1=13C
H H H
.4 Overview of 2D experiments: homonuclear shift correlated spectra via scalar couplings (DQF)COSY vicinal/geminal protons correlated COSYlr COSY optimized for small couplings (longrange couplings) INADEQUATEmostly used for carbon. carbon correlation experiments for nat. but only spins that have an additional coupling to a heteronucleus are displayed via dipolar couplings NOESY correlation via dipolar interaction ROESY rotating frame analogue of the NOESY exchange spectroscopy EXSY correlates exchanging protons (chemical and conforma tional exchange) heteronuclear shift correlated spectra via scalar coupling
Ninth Chapter: 2 dimensional NMR experiments
Pg. normal 2D experiment. Building blocks of HSQCTOCSY experiment and information content of the spectra. abundance samples.
1.LECTURE COURSE: NMR SPECTROSCOPY
1H
∆
∆
∆
∆
t1
DEC
spinlock
1H 13 C H C
∆
∆
∆
∆
spinlock
DEC
13 C H C
t1
t1
HSQC
TOCSY
HSQCTOCSY
HSQC
HSQCTOCSY
H H H
F1=13C
C A CBCC
ΩΑ ΩΑ
C AC BCC
ΩΑ ΩΒ F2=1H ΩC
ΩΑ
F2= 1H
FIGURE 11. relayCOSY COSY with additional relay step TOCSY experiment with multiple proton relay.
Ernst. 1982. Ernst. 68006801. J. O. Reson. 1976.. Magn.LECTURE COURSE: NMR SPECTROSCOPY
13
C detected experiments hetcor correlation of directly bonded protons and carbons COLOC proton. Sørensen. R. P. Ernst.. Phys. COSYLR: Bax. carbon correlation via long3 J) couplings
and carbons range (2J. W. Sørensen. U. C. Chem. R. carbon correlation via longrange (2J. R. Chem. A. 1983. Wagner. 104. R... 117. E. W.. R. 1986. 6837. Phys. M.. 85.. R. O.. O. Wüthrich. Ernst. 542561. J.
the following experiments include an additional TOCSY/NOESY/COSY transfer step: H(S)MQCTOCSY H(S)MQCNOESY H(S)MQCCOSY via dipolar couplings HOESY correlation via heteronuclear dipolar NOE) Experiments for measurement of Jcouplings homonuclear jresolved experiments heteronuclear jresolved experiments ECOSY HMQCJ
interaction
(via
1. COSYDQF: Piantini. 22292246. W.. Commun. 1981. R. G. Rance. Bodenhausen.5 Original references for 2D experiments: COSY: Aue. 479485. E. R. Biophys. G. K. Biochem.. Soc. Sørensen.. J. 3 J) couplings
1H
detected experiments HMQC. Batholdi..130
. Am. Chem. Res. Freeman. 64. J. R. W.COSY: Griesinger.
Ninth Chapter: 2 dimensional NMR experiments
Pg. 44.HSQCcorrelation of directly bonded protons HMBC proton.
1980.. 1979. Soc. 71. Bax. J.. 48494851. B.. 521528. Griffey. Soc. R. Ernst. 1984. A. 69. 1986. 65. D. J. 103.LECTURE COURSE: NMR SPECTROSCOPY
TOCSY: Bax. Reson. R. A. Magn. 1986. 4546. 185189. Summers. Bachmann. B. NMR of Proteins and Nucleic Acids. L. P. 53.. Chem. Frenkiel.. K. INADEQUATE: Bax. L. Lee. HMBC: Bax. 63. J. Chem. R. Braunschweiler. 811813. J. Soc. Chem. J. 1983. G. L. Stephens. 207213. 1980.. D. Ruben. Meier. 355360. 106. Am. Am. Reson. Lett. S. Chem. Magn. Am. Phys. A. 21022104. HSQC: Bodenhausen.. A. NOESY: Jeener. 1981. Ernst. Freeman. Davis. Reson.. Bax.131
. D. A. 108.
Ninth Chapter: 2 dimensional NMR experiments
Pg. J. Magn. 1985. 101. A. Phys. Wiley: New York. Chem.. M. Wüthrich. Müller. G. R. Am.. G. Hawkins. Soc. A. J. Am. Soc. 55. Chem. Davis. 1983. ROESY: BothnerBy. R. P.. T. J.M.. 1979. Magn. R. J. 1st ed. 301315. 1985.. 44814484. Kempsell. J. H.. A. 102. HMQC: Bax.. H. 20932094.. J. A. L. R. R. Chem. J. Freeman.. Reson. J.
We will therefore shortly review basic interactions that are important for solidstate NMR applications. For nonsp3 hybridized carbons the anisotropy can be as large as 150 ppm and even larger values are encountered for heavier elements.
Eleventh Chapter: SolidState NMR spectroscopy Pg. For nuclei with nonspherical electron distributions the shielding effect depends on the orientation of the electron cloud with respect to the static ﬁeld B0.132
.g.):
interaction nuclei
1H
typical magnitude ~15 ppm ~200 ppm ~200 ppm < 10 ppm < 140 ppm < 200 ppm ~22 kHz ~20 kHz 4. SOLID STATE NMR SPECTROSCOPY:
SolidState NMR spectroscopy is increasingly being used for a number of reasons: • • it allows to probe properties in the solid state (> material sciences) it enables to structural characterize insoluble compounds such as (synthetic) polymers it has per se no moleccular size limit and therefore is of advantage when looking at very large systems (e.1 The chemical shift It has been recognized very early on that the resonance frequency of a nucleus is inﬂuenced by the surrounding electrons. a number of additional interactions do appear that complicate spectra to an extend that they become essentially useless unless they are experimentally removed. The typical range of various interactions is listed in the table below (from Ref.5 kHz 2 kHz < 1 kHz
chemicalshift range
13C 15N 1H
anisotropy of CSA
13C 15N 1H13C 1H15N
onebond dipolar coupling
13C13C 13C15N 15N15N
1. Williamson et al. membraneproteins embedded in bilayers)
•
However. Although similar interactions are also encountered in the liquid state they are averaged out due to rapid tumbling of the molecules.LECTURE COURSE: NMR SPECTROSCOPY
1.
which reﬂects the deviation from axial symmetry:
η = ( σ xx + σ yy ) ⁄ δ
The orientation of the shift tensor is depicted below:
z
δ xx
Bo O
13
C
x
y z
δ yy
Bo
O
13
x
C
y
z
δ zz Bo
13
C
x
O
y
δ
Eleventh Chapter: SolidState NMR spectroscopy
Pg. Such a system is called the principal axis system (PAS). σyy and σzz. For the shielding tensor the zaxis points into the direction of the largest electron density.133
.LECTURE COURSE: NMR SPECTROSCOPY
The chemical shift tensor σ denotes the tensor required to transform the Bo ﬁeld vector into the vector Beff representing the effective ﬁeld:
Beff x Beff y Beff z σ xx σ xy σ xz 0 = σ yx σ yy σ yz ⊗ 0 B0 σ σ σ
zx zy zz
By suitable choice of the coordinate system the chemical shift tensor only contains the diagonal elements σxx. 3
and η the asymmetry. is rotationally invariant and corresponds to the shift observed in isotropic solution. The trace of the tensor (by convention σzz ≥ σxx≥ σyy)
σ = ( σ xx + σ yy + σ zz ) ⁄ 3
denotes the isotropic value of the shielding tensor. The parameter δ is the anisotropy and reﬂects the deviation from cubic symmetry
σ δ = σ zz – .
g.5
σxx σyy δ<0
σyy σzz
σyy σzz η=1
σzz η=0 η=0. Structural disorder or inhomogeneity represents a major issue in solidstate NMR applications!
Eleventh Chapter: SolidState NMR spectroscopy
Pg. The examples depicted at the left correspond to axially symmetric ligand ﬁelds. b) and c) microcrystalline powder obtained by evaporation of solvent with different protocols (see ref. The powder lineshapes are characteristic for the magnitudes of anisotropy δ and asymmetry η.LECTURE COURSE: NMR SPECTROSCOPY
In a noncrystalline powder sample the nuclei are randomly oriented and the observed chemical shift tensor corresponds to the statistical average over all possible orientations.5
Note that in crystalline material orientations are nonrandom! Sample preparation is very important for solidstate NMR applications:
13
C
15
N
(a)
(b)
(c)
70
60
50
40
30
20
10
140
130
120
110
100 ppm
The ﬁgure above displays 13C and 15N MAS spectra of antamanide from a) lyophilized powder. For nuclei with cubic symmetry (e.). Williamson et al.134
. 15N in NH4) even sharper signals are observed:
σxx σyy δ>0 σzz η=0 σxx
σyy σxx
σyy σzz σzz η=1
η=0.
Due to the increased linewidths sensitivity is an important issue in SSNMR.LECTURE COURSE: NMR SPECTROSCOPY
1. In the solid state no such rapid rotation exists and hence the dipolar couplings give rise to line splittings which can easily exceed 10 kHz in magnitude. Solidstate NMR experiments fundamentally suffer from the additional dipolar interactions and from the chemical shift anisotropy.
For dipolar couplings the Hamiltonian is: is homonuclear couplings
HIS = d (3 cos2β1) IZSZ . Because these dipolar couplings can be so large they are not only observed between direct neighboring nuclei but also longerreaching interactions are encountered.
d = (µo/4π) hγIγS/rIS3 d = (µo/4π) hγI2/rII3
In solution.135
.
Eleventh Chapter: SolidState NMR spectroscopy
Pg. Note that dipolar couplings have recently also been introduced into solution NMR by the use of weakly aligned systems for recording residual dipolar couplings (RDCs). For spins with I≥1. However. The scalar (J) couplings do not fundamentally differ from the quantities observed in isotropic (solution) phase.2 Dipolar couplings: Dipolar couplings depend on the
orientation of the vector that connects the two nuclei relative to the static ﬁeld. it is very important to remove heteronuclear scalar couplings to increase resolution (and hence sensitivity) in the spectra.I1I2. and for hetereonuclear couplings: HII = d/2 (3 cos2β1) 3I1ZI2Z . quadrupolar couplings exist. but those will not be described here. on the separating distance and on the
β r µA
µB
gyromagnetic ratios of the two nuclei involved. dipolar couplings are averaged to zero due to rapid (rotational) motion of the molecules (but give rise to the important relaxation phenomena). RF irradiation schemes similar to those used to remove heteronuclear couplings in solution are applied. and the quantities required are usually much larger than for solutionstate NMR.
136
. Upon rising the spinning frequencies those bands occur outside the spectral region. which describes the angular dependence in both the dipolar coupling as well as in the chemical shift Hamiltonians. The tremendous linenarrowing effects due to MAS are displayed in the powder 13C spectra of 13C.4 Sensitivity Enhancement: Solutionstate NMR normally utilizes proton detection experiments due to the high gyromagnetic ratio of protons which makes this nucleus very sensitive. is a much less sensitive nucleus. and mostly carbon detection is used instead. 1.15N labeled glycine below (from ref.74° (magicangle spinning. The broad lines observed in the powder spectrum collapse into rather sharp lines which occur at the position of the isotropic chemical shift.3 Magic Angle Spinning (MAS) The effects from chemical shift anisotropy and dipolar couplings lead to excessively broad lines.7° ωr
5 kHz
10 kHz
270
240
210
180
150
120
90
60
30
0
30
δ [ppm]
At the lower frequencies spinning sidebands fall into the spectral regions of interest. To a large extend this linebroadening can be removed by spinning the sample about the magic angle 54. For this angle the secondorder legendre polynomial (3cos2β1)/2. In MAS.LECTURE COURSE: NMR SPECTROSCOPY
1. Williamson et al. however.):
static
B0
θ m = 54. vanishes. Polarizationtransfer techniques are therefore employed to
Eleventh Chapter: SolidState NMR spectroscopy
Pg. MAS). In SSNMR applications proton detection is less favorable due to the large number of homonuclear dipolar interactions. Carbon. Unfortunately spinning the samples at high frequencies does heat up the sample (tens of degrees!) and the heating effect must be removed by cooling the rotor appropriately. the sample is placed in a cylindrical rotor which is rapidly spun (up to 3050 kHz) about an axis that is tilted by 54° away from the direction of the static ﬁeld.
Due to the fourfold higher γ of protons the energy difference between α and βstates is much larger for protons and hence the population difference is also larger.1 Nowadays HartmannHahn Cross Polarization (CP) is used. This technique may therefore be use to derive distance information of pairs of nuclei:
Distance in Å
1.LECTURE COURSE: NMR SPECTROSCOPY
transfer the much more favorable proton polarization onto the carbon nuclei. The extend by which magnetization can be transferred to the heteronucleus by HartmannHahn cross polarization transfer depends on the mixing time.
Eleventh Chapter: SolidState NMR spectroscopy Pg.5
3. The basic idea behind recoupling techniques is that the averaging of spatialdependent parameter such as dipolar couplings or chemical shift anisotropy under MAS can be removed if RF pulses are applied in rotorsynchronized fash1.5
3 6
Mixing time [ms]
Fig. One way to yield information on spininteractions is to spin the sample more slowly and extract spin interaction parameters from spinning sideband amplitudes. A two spin system has been used and interactions other than dipolar couplings are neglected. and advantage can be taken from the much shorter T1 of protons (higher repetition rate). A more commonly technique used nowadays is to reintroduce spininteractions by recoupling techniques. In this technique B1 ﬁelds are simultaneously applied to the proton and carbon spins such that the precession frequencies of the two spins are approximately equal (γΗBH ≈ γCBC). but also on the distance between the nuclei. Right: Signal buildup curves for various durations of mixing time and distances.5
I S
2. the removed interactions need to be reintroduced in order to yield distance or (dihedral) angle information. Left: Pulse sequence for hetero CP.5 Recoupling techniques in SSNMR: Whereas magicangle spinning (MAS) leads to signiﬁcant linenarrowing in 1D spectra. Thereby. carbon polarization can be enhanced by up to a factor of four (γΗ/γC).
1.137
.
: a) REDOR pulse sequence for recording 13C. 15N.2 0
0
1
2
3
4
5
6
dephasing period (ms)
Fig.)
Eleventh Chapter: SolidState NMR spectroscopy
Pg. Note that during signal acquisition the dipolar couplings are removed by MAS so that narrow signals are sampled:
a)
1H 13C
π/2
CP CP
decoupling
15N
0 1 2 3 4 5 6 7 8
t/τr
b)
1 0.LECTURE COURSE: NMR SPECTROSCOPY
ion: HIS
ωr
S= 0
ωr
(
1/2ωr
1/2ωr
)n
S 0
HIS For heteronuclear dipolar recoupling rotational echo double resonance (REDOR) has been used. Herein. b) Relative signal intensity change against mixing time for a onebond dipolar coupling (from ref.8
∆S 0.15N dipolar couplings.13C dipolar couplings are reintroduced during the mixing time leading to dephasing of the signal by the dipolar coupling.138
.4 0.6 S0
0. Williamson et al.
Eleventh Chapter: SolidState NMR spectroscopy
Pg.2 0. Note the change in signal amplitude of the signal at 53 ppm after the resonance at 20 ppm has been selectively inverted.: a) Pulse sequence for a rotational resonance experiment. which in turn depend on their distance. c) Change in intensity vs.139
. b) 1D spectra taken at various setting of the mixing time τm.8 0. The dipolar coupling is then measured in form of an exchange experiment: A resonance A is selectively inverted and the exchange between the two sites is monitored:
a)
π/2
ωr = 3411Hz
b)
CP
π/2
Decoupling
π/2
25ms
20ms
CP
Cross DANTE Polarization Inversion
τm
15ms
c)
1. it is complicated by the fact that the spininteractions need to be reintroduced selectively. a reference spectrum without the rotorsynchronized 15N pulses is additionally acquired and the intensities in both spectra are compared. in rotational resonance (R2) the rotor frequency is set to a submultiple of the chemical shift difference. ωdq=ωa plus ωb).4 0. Whereas its implementation is similar to the heteronuclear variants in principle. Homonuclear recoupling techniques have also been developed. mixing time. The curves reﬂecting signal intensity versus time depend on the magnitude of the heteronuclear dipolar couplings. (A transformation IzSz > Iz(Sz) is easily accomplished for a heteronuclear pair of spin by just applying a 180° pulse to the S spin but usually difﬁcult for homonuclear spin pairs which will both be ﬂipped unless the 180° pulse is selective).0
<I1zI2z>
5ms
0ms
0
10 τm (ms)
20
30
70
60
50
40 δ (ppm)
30
20
10
Fig. For example.6 0.or doublequantum frequencies of the spin pair of interest (ωzq= ωaωb. Note that strictly speaking the RF pulses are applied to the spinpart of the dipolar Hamiltonian (IzSz) whereas MAS only inﬂuences the spatial part (3cos2θ1). and precise values for these can be extracted from the data by comparison of the experimental data with simulations.LECTURE COURSE: NMR SPECTROSCOPY
Usually.0
10ms
0. Selectivity in the homonuclear recoupling techniques is achieved by adjusting a chosen property such as the spinning (rotor) frequency to the zero.
can be oriented either by deposition on glass plates or by magnetic alignment. Other techniques use double quantum transitions (HORROR. all other interactions need to be sufﬁciently eliminated. 1. DREAM. however.LECTURE COURSE: NMR SPECTROSCOPY
The condition for rotational resonance is fulﬁlled only for one (or at least a very few) spin pairs.). Opella et al. In addition. It has recently become clear that the orientation of helices with respect to the membrane normal may be probed from a combination of chemical shift and the onebond dipolar 15N. and lipid bilayers oriented on glass plates (B) (taken from ref.g. Therefore. e. magneticallyoriented lipid bicelles (A).140
. DRAMA etc. crystalline materials or peptides/proteins embedded in phospholipid membranes. methods relying on scalar couplings are rather new and their use has been motivated by the fact that they allow to trace along covalent networks (spin systems) leaving less ambiguity in the assignment process.6 SSNMR of oriented samples: SSNMR spectra are complicated by the fact that all kind of orientations with respect to the external ﬁeld are possible. Some classes of molecules. and largely utilize rotorsynchronized RF pulses.). Assuming a unique association with the membrane the nuclei therefore do not adopt all possible orientations with respect to the external ﬁeld. Jcouplings are much smaller and transfer times are much longer. This effect is demonstrated in the ﬁgure displayed on the left: The spectra were recorded on 15N fd coat protein on unoriented lipid bilayers (C).1H couplings in the socalled PISEMA spectra:
Eleventh Chapter: SolidState NMR spectroscopy
Pg.
In addition. Peaks belonging to amide moieties from a single helix in PISEMA spectra occur at characteristic places.141
. the orientation of axes in the PAS are depicted.LECTURE COURSE: NMR SPECTROSCOPY
. A) displays peak position for various angles the helix makes with
Eleventh Chapter: SolidState NMR spectroscopy
Pg. and the exact position and pattern can be rather easily related to the angle the helix makes with the membrane surface (and hence with the ﬁeld):
A
B
β
C
In the ﬁgure above.
σ
O
Βο
22
C Cα−1
σ
Cα Ν θ=17 ο H
11
σ33
220 140 σ{15Ν}
The ﬁgure above displays the chemical shift of a 15N amide resonance in dependence of the orientation of the helix.
S. Meth. Tycko. in which NMR active isotopes (13C. Rev. biosynthetic routes to prepare alternatingly labeled peptides (only every second position is labeled) or selectively at methyl position have been developed. B. (2001). In C) typical PISEMA spectra are displayed for the AchR M2 peptide (right) together with the HSQC spectrum recorded in solution on the DPCmicellebound form. Assigning polypeptides from uniformly labeled material still presents a major challenge.M. SolidState Spectroscopic Methods in Chemistry. R. (2002). For that purpose a series of tripleresonance experiments relying on transfer pathways similar to those used for solution studies have been suggested. Whereas selectively labeled small molecules usually need to be synthesized from suitable precursors. Angew.15N) are only introduced at speciﬁc sites. Zerbe.F. M. Biomolecular Solid State NMR: Advances in Structural Methodology and Applications to Peptide and Protein Fibrils.J. Ma.D. H.
•
•
•
Eleventh Chapter: SolidState NMR spectroscopy
Pg. C. Annu. 285313. Hence. Williamson. Wiley 2003. O. F. A. D. Nuclear Magnetic Resonance of MembraneAssociated Peptides and Proteins. 52. 1. peptides can be subsequently labeled at cysteine sites with methyliodide. Ernst.142
. Alternatively.LECTURE COURSE: NMR SPECTROSCOPY
the membranenormal. 575606.
References: • P.T. Jerschow. Bitter. Phys. Chem. Enzymol. 30963129. microcystalline solids are usually required and much attention needs to be paid to sample preparation. Another common problem is that signals are inherently broadened due to the structural disordered in noncrystalline solids. Marassi. In B) experimental peak positions are assigned to the helical wheel positions for 15N labeled AchR M2 peptide (a channelforming peptide) in oriented bilayers and compared to a simulated PISEMA spectrum calculated for a tilt angle of 12°. Ed. 339. Chem.H. 41. Opella. Meier: MAS SolidState NMR of Isotropically Enriched Biological Samples in BioNMR in Drug Research.M. (2001).7 Labeling strategies for solidstate NMR applications: The easiest (and most often used) remedy to reduce the vast number of interactions in the solid state is the use of selectively labeled material. Laws.