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NMR Spectroscopy
Gottfried Otting
- shim
- tuning/matching
- pulse calibration
2D NMR
- the 5 truly important 2D experiments (small molecules)
11
13
22
24
27
1
- Phase correction
31
- Window multiplication
36
- Zero filling
41
- Baseline correction
42
Understanding 2D NMR
- The NOESY experiment
44
- Phase cycling
49
49
- t1-noise
54
- Sensitivity of 2D NMR
55
56
60
62
- Spin-echo
63
65
66
68
- Reverse INEPT
69
- Broadband decoupling
70
71
DQF-COSY
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76
79
80
83
85
Selective pulses
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89
Lock:
The magnetic field of the spectrometer has to be extraordinarily stable, because
the frequency differences between the nuclei are so small.
Problem 1: a) Assume that a spectral resolution of 0.4 Hz is adequate. How many
ppm are 0.4 Hz on a 400 MHz NMR spectrometer?
b) To put this in perspective: if Mt Everest is about 10,000 m high,
what height is 0.1 ppm of Mt Everest?
NMR magnets need to stand clear of magnetic perturbations (any magnetic tools,
cars parked outside the NMR room, lifts etc.) Even a small thermal expansion of
the outside of the magnet by a change in temperature (worst case: the sun shining
on the magnet) causes a noticeable change in the magnetic field.
adjusts the magnetic field strength by increasing or decreasing the current through
a helper magnetic coil, which is positioned at the very inside of the magnet in the
room temperature section.
The lock signal usually only reports the height of the (strongest) peak found in the
2
H NMR spectrum.
Problem 2: An external lock is a small vial containing a deuterated solvent
built into the probe head, obviating the need for using
deuterated solvent for the NMR measurement. What then is the
point of using deuterated solvents?
If the spectrometer knows which solvent it is locking on, it can use this for
spectrum calibration!
Shimming:
While the lock can make the magnetic field stable, the field also needs to be
homogeneous, i.e. the same across the entire sample. This is achieved by
numerous shim coils in the room temperature section of the magnet (room
temperature shims).
The axis of the magnet is defined as the z-direction. Changing the z-shim means
that the magnetic field is made stronger at one end of the sample than at the other,
with a linear gradient along the z-axis.
For the z2-shim, the gradient is quadratic (like a parabola).
There are z3, z4, z5, z6, z7, z8 shims
The shims are designed to be orthogonal to each other, but in practice changing
one will affect others as well.
On-axis shims or spinning shims are the shims z z8.
Off-axis shims or non-spinning shims are all shims that contain x and y
components (e.g. x, y, xy, x2-y2, xyz, xz, yz2, etc.)
Problem 3: a) In which direction does the magnetic field point that is generated by the
x-shim?
b) In which direction does the x-shim change the magnetic field gradient?
to spinning sidebands in the NMR spectrum). For good off-axis shims, the lock
signal does not drop very much when spinning is turned off.
!
Fortunately, shimming has been automated with the help of pulsed field gradients
(PFG, to be discussed later).
For a good shim, the 1H NMR line width of CHCl3 is < 8 Hz at the height of
the 13C satellites (without spinning).
Note: taking a sample out of the magnet and re-inserting it into the magnet
changes its position - good idea to shim the on-axis shims again!
90 degree pulse:
The biggest signal in the NMR spectrum is obtained after a 90o pulse (if only a
single pulse is applied). The 90o pulse length, delivered with the same power,
depends on the solvent. In particular, 90o pulses are longer for salty samples. If an
experiment requires an exact 90o pulse, it needs to be determined experimentally.
2D NMR
Tabulated chemical shifts and coupling constants are not enough:
- Chem. shifts depend on solvents, temperature, pH etc.
- Coupling constants are difficult to resolve under conditions of spectral overlap
and strong coupling (established data bases often refer to spectra recorded on
low-field NMR spectrometers)
Not much beats the clarity of an assigned [1H,13C]-HSQC spectrum!
Not to mention the many parameters that are accessible only by 2D NMR
11
Albert
W. Overhauser
!
1925-2011
12
- The cross-peaks correlate the 13C spins with the directly bonded 1H spins.
- As in most 2D NMR spectra, when plotting the 1D NMR spectra along both
axes the cross-peaks appear at the intersection between peaks in the 1D
13
C-
13
14
15
16
17
- The cross-peaks correlate the 1H spins via one or multiple JHH couplings
within a spin-system.
- The cross-peaks appear at the intersection between peaks in the 1D 1H-NMR
spectra.
- Cross-peaks tend to be positive, but on closer inspection reveal impure line
shapes.
- The spectrum is symmetric about the diagonal.
18
- A cross-peak is weak (or absent) if too many JHH couplings are required to
link the two 1H spins, or if one of the JHH couplings in the chain is very small.
A spin system is a set of 1H spins that are all directly or indirectly linked by
JHH couplings. Example: ethylbenzene. The aromatic ring presents one spin
system. If no JHH coupling can be observed between the ethyl group and the
phenyl ring, the ethyl group presents a second spin system.
19
20
The NMR spectra of menthol are actually trickier to assign than one might think
for such a small molecule. Note, however, the excellent resolution in the
13
C-
HSQC spectrum: all cross-peaks are nicely separated, providing a MUCH better
fingerprint of the compound than a 1D 1H-NMR spectrum! So, its worth
assigning the 13C-HSQC spectrum.
21
Quadrature detection
First, each data point in the FID actually comes from two measurements that are
combined into a complex number. The real part contains one measurement, the
imaginary part contains the other measurement. The two measurements are of the
transverse magnetisation, measured as the projection onto the x and y axis,
respectively, in the rotating frame (more about this below).
22
By measuring the projections onto both the x and y axes, we can accurately tell,
which way the magnetisation vector points. Collect a second complex data point a
short time (dwell time) later and we know the sense of precession (clockwise or
anti-clockwise).
23
How can an NMR spectrometer collect complex data points with a single coil
The NMR spectrometer detects the precessing magnetisation by a single coil, i.e.
the signal is indistinguishable from a linearly polarized magnetisation oscillating
at the Larmor frequency (400 MHz, if the spectrometer is a 400 MHz NMR
spectrometer).
Footnote: physicists established that = -B0, i.e. the Larmor frequency is
negative for nuclear spins with positive gyromagnetic ratio (most spins,
such as 1H,
13
C,
19
F,
31
For good measure, 15N has a negative , but chemists always plot 15N-NMR
spectra still with frequencies (and ppm values) increasing from right to left
NMR spectroscopists simply dont care about the sign of . On a 400 MHz
NMR spectrometer (as in the figure), we just care about the 1H NMR
spectrum found in a 10 ppm window!
24
25
Low-pass filters remove the sum frequencies (about 800 MHz) and let the
audiofrequencies = 0 pass.
Each of the two signals, modulated by cos(t) and sin(t), are then digitised by
analogue-to-digital converters (ADC), which forward the results to the
computer.
26
Fourier transformation
Mr Fourier claimed that he could decompose any function into a sum of sine and
cosine functions. Fourier transformation simply means a plot of the amplitudes of
the requisite sine and cosine functions (which have, of course, different
frequencies) versus the frequency.
Actually, two plots. One is for the cosine functions, the other for the sine
functions. We call one the real part, the other the imaginary part. Making both
plots is referred to as complex FT.
Fourier transforms are straightforward to calculate: multiply the FID with a cosine
function of a specific frequency (a test cosine function, if you like) and calculate
27
the integral. If the integral is zero, this particular cosine function is not contained
in the FID.
Remember, the integral of a cosine function is zero (there are as many positive as
negative areas in the function). The integral of a squared cosine function,
however, is finite. So, if our test cosine function is in any way present in the FID,
the integral will be non-zero. For test cosine functions with the wrong frequency,
the integral will be zero. Fouriers idea was to test the FID with cosine functions
that systematically increase in frequency, then plot the integral values found
against the frequencies tested. Once its been done with cosine functions to get
the real part, it can be repeated with sine functions to get the imaginary part.
Voil!
The FT transforms the FID from the time domain to the frequency domain.
28
A minor complication:
The FID is complex:
The FT is also complex (real part produced by a cos-FT, imaginary part produced
by a sin-FT). In effect, this situation produces 4 FTs: a cos-FT and a sin-FT of the
real part of the FID, and a cos-FT and a sin-FT of the imaginary part of the FID.
It turns out that adding the result of the cos-FT of the cosine-modulated signal to
the result of the sin-FT of the sine-modulated signal yields sign discrimination,
i.e. we can tell whether the frequency of the NMR signal relative to the
spectrometer frequency was positive or negative. (The above applies to the real
part of the Fourier transformed spectrum. For the imaginary part, we add the
result of the sin-FT of the cosine-modulated signal to the result of the cos-FT of
the sine-modulated signal.)
29
The bubbles highlight the real part, the remainder is the imaginary part. Note how
the imaginary number i = Sqrt(-1) neatly arranges for the right combinations of
the results of the 4 FTs. Only the real part is displayed on the computer
screen. The imaginary part is needed for phase correction.
30
Phase correction
The FT of an exponentially decaying signal S(t) = cos(t).exp(-t/T2) is a
Lorentzian. The cos-FT of a cosine-modulated signal delivers an absorptive
Lorentzian, whereas the sin-FT of the same signal delivers a dispersive
Lorentzian:
31
One has to be very lucky to find a purely cosine-modulated signal in the real part
of the FID, so that the real part of the FT delivers a purely absorptive spectrum.
All usual cases require a phase correction after the FT to produce a spectrum with
absorptive lineshapes.
32
33
Phase correction is simply a linear combination of the data points in the real and
imaginary part of the spectrum:
S() = exp(icorr)S0()
where S0() is the complex spectrum (i.e. both real and imaginary part) before
phase correction and corr is the phase of the correction. The equation defines the
zero-order phase correction.
Problem 10: Use exp(i) = cos + isin to show that the new real part of the spectrum
becomes after phase correction SRe() = cos(corr)S0Re() - sin(corr)S0Im()
To find the optimal phase, NMR software allows interactive phase correction.
Sometimes one needs a frequency dependent phase correction or so-called firstorder phase correction. The illustration below is again from the book
Understanding NMR Spectroscopy by James Keeler.
34
First-order phase corrections become necessary, when the magnetisation had the
chance to precess for a short while before the first point of the FID is recorded.
35
Window multiplication
The quality of a NMR spectrum can be greatly enhanced by multiplying the FID
with a so-called window function prior to FT. A window function is a function
that starts where the FID starts and ends where the FID ends. Quite trivial.
Problem 11: Show that, if the FID is multiplied by an exponential function exp(-Lt),
the resulting linewidth (FWHH) after FT will be broader by L Hz.
Hint: check out the solution to problem 9.
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37
38
Window multiplications can also be used to make the lines in the spectrum
narrower. This can be achieved by
using Gaussian multiplication. This
function attenuates the FID at the
beginning, emphasising its later parts.
The function has two parameters. GB
determines the position of the maximum as a fraction of the acquisition time (0 <
GB <1). LB is set to about -0.5 times the natural linewidth at half height. S/N
drops and integrals become less reliable.
The first point of the FID is proportional to the total integral in the frequency
domain. A window function that decreases the first data point of the FID relative
to later ones leads to negative side lobes for each peak in the spectrum. In this
situation, correct integrals are obtained only if the negative side lobes are
included in the integral measurement.
39
The point of this function is to bring the end of the FID smoothly to zero. This is
important if recording of the FID ended before it disappeared in the noise. The
effect of truncation is well illustrated in Keelers book Understanding NMR:
Sometimes multiplication by the cosine window still leaves small wiggles behind.
In this case, try the squared cosine window.
Problem 12: a) What does the squared cosine window look like?
b) Which window function generates more linebroadening, the cosine or
the squared cosine window?
40
41
because it can be reconstructed at any time from the real part by a so-called
Hilbert transform.
Baseline correction
Measuring integrals will be problematic, if the baseline has a non-zero offset,
slope or bend like a washing line. This can be fixed by subtracting a function that
follows the baseline. This can be done by fitting a polynomial (y = a + bx + cx2 +
dx3 + ) manually or automatically. Manual corrections solve the problem that
automatic routines may have with distinguishing very broad peaks from baseline.
42
Understanding 2D NMR
The first 2D NMR experiment (2-pulse COSY) was proposed 1971 by the Belgian
physicist Jean Jeneer at an AMPERE summer school in former Jugoslavia.
Richard R. Ernst received the Nobel Prize in Chemistry in 1991 for the
development of FT NMR and 2D NMR.
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44
The figure below explains the NOESY pulse sequence with magnetisation vectors
for two spins, A (blue) and B (red). The three 90o pulses are delivered with the
phases 1, 2 and 3, respectively. By default, 1 = 2 = 3 = x. For simplicity, we
start with A-spin magnetisation only.
45
46
b) Draw the right-handed Cartesian coordinate system: z-axis up, y-axis to the
right, x-axis coming out of the plane towards you.
47
4) The 2nd 90ox pulse converts the magnetisation in the x-y plane into
magnetisation in the x-z plane.
5) Only the z-component of the magnetisation after the 2nd 90ox pulse is retained
(achieved by phase cycling, explained below), i.e. any transverse
magnetisation components are discarded. During the mixing time m, part of
the A-spin magnetisation (blue) is transferred to the B-spin (red) by NOE.
6) The 3rd 90ox pulse turns z-magnetisation into y-magnetisation, which
generates the FID during the detection period t2.
After each scan, a recovery delay is required to re-establish equilibrium
magnetisation (by T1 relaxation) before the next scan.
48
Phase cycling:
Phase cycling involves the summation/subtraction of scans recorded with different
phases of the pulses. (All other parameters are kept the same.) In our example, the
phase of the 3rd 90o pulse is changed from x to x to obtain a second scan, where
the magnetisation of interest points along the y-axis rather than the y-axis. By
subtracting the result of this second scan from that of the first scan (which was
recorded with a 90ox pulse rather than a 90o-x pulse), the signals add up.
In contrast, this phase cycle subtracts any x-component of the magnetisation
present at the end of m (because x-magnetisation is unaffected by the rotation
around the x-axis and, hence, doesnt change its sign).
Any y-component of the magnetisation present at the end of m is turned into
longitudinal magnetisation by the 90ox pulse. Longitudinal magnetisation does not
precess and therefore does not induce a current in the coil (i.e. it is not detected).
49
the FIDs look as shown below (after FT(t2 -> 2), i.e. after transforming each FID
recorded with different t1 delay into a spectrum, i.e. converting the time domain t2
into the frequency domain 2.
The result of the FT(t2 -> 2) transformation is illustrated above for four different
FIDs recorded with different t1-times:
50
i) t1= 0. The spectrum contains a peak for the A-spins and a smaller one for the Bspins (which originates from the NOE during m).
ii) t1 so that At1 = /2, i.e. the A-spin magnetisation precessed by precisely 90o
during t1. No magnetisation is observable, because x-magnetisation present at
the end of t1 remains x magnetisation after the 2nd 90ox pulse and, hence, is
destroyed by phase cycling.
iii) t1 so that At1 = , i.e. the A-spin magnetisation precessed by precisely 180o
during t1. The A spin magnetisation simply changes sign by the end of t1.
Therefore, all signals resulting from this magnetisation are inverted.
iv) t1 so that At1 = 3/2, i.e. the A-spin magnetisation precessed by precisely
270o during t1. This turns it into magnetisation aligned with the x-axis at the
end of t1 which is destroyed by phase cycling.
Problem 14: In the figure above, why does the peak amplitude of the B-spin oscillate
with the frequency A? Hint: consider the origin of the B-spin
magnetisation.
Clearly, the t1-2 data matrix contains signals at the 2 frequencies of the A-spin
and the B-spin which oscillate in sign and magnitude as a function of t1 with the
frequency A. Looking along the t1 axis, the signal intensities observed at 2 = A
51
are modulated like a damped cosine function. The cosine function arises from the
fact that of the magnetisation precessing during t1, only the projection onto the yaxis is selected. The dampening arises from T2 relaxation during t1. The signal of
the B-spin oscillates with the same frequency as the signal of the A-spin (namely
A).
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the likelihood of magnetisation transfer from A-spins to B-spins is the same as for
that from B-spins to A-spins.
Problem 15: Where in the spectrum will the cross-peak be that originates from B-spin
magnetisation at the start of the NOESY experiment?
53
t1-noise:
What happens if the magnetic field or frequency of the electronics is not perfectly
stable during a 2D NMR experiment? If the spectrum randomly shifts a little in
frequency from FID to FID, or its amplitude changes slightly, the traces taken
along the t1 axis of the 2D data matrix will become noisy. The Fourier transform
of noise is noise. Hence, noise bands appear in the F1 dimension. To minimize t1noise, the room temperature should be stable to within 0.5 degrees and no
bicycles, trolleys, cars must come anywhere near the magnet.
Problem 16: a) Why are the F1 and F2 dimensions often referred to as the indirect and
direct dimensions, respectively?
b) Why is there no such thing as t2-noise?
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Sensitivity of 2D NMR:
For a single scan, a good signal-to-noise (S/N) ratio is obtained by recording the
signal until it starts to disappear in the noise.
In a two-dimensional NMR experiment, every point of the 2D data matrix
contributes to the S/N ratio of the final two-dimensional peak. As a 2D NOESY
spectrum is typically recorded with about 512 data points in the indirect
dimension, resonances become observable that are below the level of noise in
every single FID of the NOESY experiment.
55
The maximal acquisition times in the t1 and t2 dimensions are called t1max and t2max,
where t2max is simply the acquisition time of each FID. As we need to wait for the
recovery of equilibrium magnetisation between scans anyway, we can use the
entire recovery delay for acquisition without making the 2D experiment any
longer.
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Doubling t1max, however, doubles the duration of the 2D NMR experiment! With
the next NMR user breathing down your neck, you may decide that some lesser
resolution in the indirect dimension is perfectly acceptable
57
Problem 17: Why are NOESY spectra symmetric about the diagonal in principle, but
the cross-peaks appear elongated in the F1 dimension in practice?
Harry Nyquist
1889 - 1976
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outside the spectral width, it will still appear in the spectrum, but at the wrong
frequency. This effect is called folding or aliasing.
The picture shows, how the red signal will falsely appear to have the
frequency of the blue signal, if sampled at the blue time points.
The position of the folded signals can, however, be predicted. There is also a
recipe for folding in such a way that the folded signals acquire the opposite
sign. The diagonal of a 2D NOESY spectrum appears like this:
2) Use the original t1-increment and sample up to t1max, but omit some of the FIDs
on the way. Then go on to reconstruct the missing points from the recorded data
points. Then Fourier transform the repaired data set as usual.
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This method has become the topic of active research in the past few years, as
computers have become sufficiently fast to perform the reconstruction in
reasonable time. It works fine so long as the number of experimentally recorded
FIDs comfortably exceeds the number of peaks in each F1 cross-section.
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Problem 18: The function sin(At1) is zero for t1 = 0 and maximal for t1 = /2. Using
the vector description of the NOESY experiment, show that 1 = y and 2
= x results in amplitude modulation of the FIDs that is phase-shifted by
90o relative to the situation where 1 = 2 = x.
The upshot is that, omitting some of the FIDs to save time is fine, but each t1data point still needs to be recorded as a complex data point, i.e. with the two
different phase settings of 1 and 2.
61
Geoffrey
Bodenhausen
*1951
cross-peaks with purely absorptive lineshapes that are singlets in the indirect
62
13
C-HSQC spectra are the most sensitive way of measuring 13C-chemical shifts!
The HSQC experiment encompasses concepts that are important in many NMR
experiments: spin-echo, in-phase and antiphase magnetisation, magnetisation
transfer through scalar couplings, refocussing of heteronuclear couplings,
broadband decoupling.
Spin-echo:
The spin-echo sequence is (t 180o t). It refocusses chemical shifts, but the Jcouplings continue to evolve during the entire duration 2t.
Consider chemical shift evolution first. The figure below illustrates the
refocussing effect for the three different spins f, i and s:
At the end of the spin-echo, all magnetisation vectors are again aligned, as if no
chemical shift evolution had occurred.
Problem 19: Show that a 180oy instead of a 180ox pulse would also refocus the spins.
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Now consider J-coupling evolution for a simple doublet (ignoring chem. shift
evolution). We describe the doublet by two vectors (blue and red) precessing with
slightly different frequencies.
Remember: doublets arise, because the coupling partner can be in the state or .
This happens to the doublet in the spin-echo (note: vectors depict the
magnetisation of only a single doublet, the doublet of the coupling partner of
course exists but is not shown):
So, in the case of the doublet in a spin-echo, the 180oy pulse appears to do
nothing! This is the combined result from two effects of the 180o pulse: (a)
flipping the magnetisation vectors by 180o about the y-axis and (b) inverting
longitudinal magnetisation (equivalent to interconverting the and states of the
coupling partner), which amounts to repainting the red vector blue and the blue
vector red.
The upshot is that the spin-echo always refocuses the chemical shifts, but not the
J-couplings so long as the 180o pulse hits the coupling partner as well.
64
During
free
precession,
in-phase
magnetisation
converts
to
antiphase
Left panel: as before (the two doublet components are shown with different
colours)
Centre: uses a different colour code: the two doublet components are both painted
red (as they belong to the same type of spins (e.g. spin A), just located in different
66
molecules. The spin state of the (blue) coupling partner is in the molecules for
which the vector points along the positive y-axis, and in the molecules for which
the vector points along the negative y-axis.
Right panel: same as centre panel, except that its a perspective drawing and the
spin states of the blue coupling partner are indicated by vectors along the z-axis.
From the representation of the right panel one would expect that a 90ox pulse
would convert antiphase magnetisation of the red spin into antiphase
magnetisation of the blue spin. This is indeed the case!
Magnetisation transfer via scalar couplings:
A 90o pulse applied to both spins converts magnetisation of spin A (that is
antiphase with respect to spin B) into magnetisation of B (that is antiphase with
respect to spin A).
antiphase
magnetisation
to
antiphase
13
C-
67
Advanced Problem II: Why is it necessary that the phase of the second 90o(1H) pulse
in the HSQC pulse sequence is y (and not x)? Hint: this pulse must
convert transverse 1H vectors into longitudinal (z) vectors.
13
through, the spin-states of 1H are inverted. Therefore, all JHC couplings are
refocused by the end of t1 and only the chemical shift evolution of 13C dephases
the magnetisation.
68
In this sequence, the chemical shift evolution is refocused for 13C (but not for 1H).
All JHC couplings are refocused because the 180o pulse does not invert the spin
states of the 1H spins; its a simple spin-echo for 13C spins, irrespective of whether
the different
13
peak splittings due to JHC. This explains, why the INEPT experiment needs 180o
pulses on both 1H and 13C spins! (Otherwise the JHC couplings would be refocused
to in-phase magnetisation, which cannot be transferred to the heteronucleus.)
Reverse INEPT:
The last part of the HSQC pulse sequence is called reverse
INEPT because it looks like the initial part in reverse.
The 90o(1H,13C) pulses at the start of the reverse INEPT
convert transverse 13C-magnetisation (antiphase with respect
to 1H) back to 1H-magnetisation (antiphase with respect to
13
C).
13
13
The 90ox(13C) pulse of the reverse INEPT changes y- but not x-magnetisation.
How does this produce peak amplitudes modulated by cos(Ct1)?
69
Broadband decoupling:
During data acquisition (t2), each 1H-resonance would be split into a doublet by
1
the 13C-spin states rapidly between and . The decoupled signal appears in the
centre of the doublet.
Problem 21: Too much pulse power during broadband decoupling fries the sample,
amplifiers and probehead. Use the condition for fast chemical exchange k
>> A - B to estimate a rate with which the 180o pulses must be delivered
for decoupling!
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The delay 1/(2JHC) is tuned to small 2J and 3J couplings. During this delay, both
chemical shifts and couplings evolve. As a result of the chemical shift evolution,
the phases of the magnetisation detected during t2 are messy (a mixture of
absorption and dispersion that depends on the Larmor frequency of the
resonance). The F2 dimension is often presented in magnitude mode: I = Sqrt(IRe2
+ IIm2), where IRe is the signal in the real part of the spectrum and IIm is the signal
in the imaginary part of the spectrum.
The 13C-dimension is relatively clean. JHC couplings are refocused during t1, but
JHH couplings evolve (and result in correspondingly broader peaks in the F1
dimension).
Problem 23: a) Why are JCC couplings during t1 not a problem at natural isotope
abundance?
b) Why is decoupling during acquisition not a good idea in the HMBC
experiment?
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Cross-peaks due to 1JHC couplings are not welcome in HMBC spectra. They are
usually suppressed by a 90o(13C) pulse combined with suitable phase cycling to
kill all antiphase magnetisation present after the delay 1/(21JHC):
Note that this pulse sequence is not much longer than the one before, because
1/(21JHC) is much shorter compared with 1/(2nJHC) (n > 1).
Problem 24: If you suspect the nJHC coupling to be really small, which delay could you
adjust to have a better chance of observing a cross-peak?
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DQF-COSY
Below is the DQF-COSY spectrum of sucrose from the library generated by
Teodor Parella at http://triton.iqfr.csic.es/guide/manualw.html
The spectrum is symmetric about the diagonal. Each cross-peak has an equal
number of positive and negative multiplet components.
Cross-peaks have antiphase lineshapes for the active couplings (i.e. the coupling
generating the cross-peak) and in-phase splittings for the passive couplings (i.e.
any couplings with third spins).
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The insert demonstrates that cross-peaks can appear only at intersections where
there are signals in the 1D NMR spectra. No signal in the 1D, no cross-peak!
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The zoom shows that proton 2 couples with 3, 3 couples with 4, etc.
The magnitude of the active couplings can be estimated from the separation
between the antiphase components.
The diagonal peaks are not purely absorptive (hence, use the cross-peaks for
phase correction!)
75
76
The maxima of the two Lorentzian lines composing the antiphase doublet are
closer than the maxima of the resulting antiphase doublet (dashed lines). The
cancellation of peak intensity in the centre of the antiphase doublet means that it
has overall smaller peak heights and the apparent coupling constant is too large.
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In fact, when the coupling constant is significantly smaller than the line width, the
same splitting is observed irrespective of the actual coupling constant:
Problem 25: Imagine an in-phase doublet with a fixed coupling constant J. In a thought
experiment, allow the line width LW to increase gradually, going from
LW << J until LW = J. Will the peak separation remain the same?
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The NOE from spin A to spin B is positive, the relayed NOE from A to C (via B)
is negative and the double-relay (A to D via B and C) is again positive.
Problem 26: (a) How would T1 relaxation during the mixing time change the NOE
build-up curves?
(b) How can one distinguish direct NOEs from spin-diffusion?
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CH3-C(O)-N(CH3)2
2.0
D1(1H)/ppm
2.5
2.0
2.5
D2(1H)/ppm
2.5
2.0
D2(1H)/ppm
The spectrum on the right is the NOESY spectrum with a mixing time m = 60 ms,
plotted at fairly high contour levels to highlight the different peak heights of the
cross-peaks and diagonal peaks. If m is short, the exchange rate kex of the methyl
groups can easily be calculated:
kex = (IC/ID)/m
where IC is the intensity (volume!) of the cross-peak and ID the intensity of the
diagonal peak. (Strictly speaking, ID should be measured in a spectrum with zero
mixing time. The equation is valid only if T1 relaxation and spin-diffusion during
the mixing time can be neglected, which is OK for short mixing times.)
The spectrum on the left was recorded with m = 4 s (!). The chemical exchange
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has equilibrated the intensities of the diagonal and cross-peaks. The NOE crosspeaks with the acetyl group are negative (and much weaker very low contour
levels had to be plotted to make the NOEs visible).
Problem 27: Why are the NOEs between the acetyl and amide methyl-groups of the
same intensity after a mixing time of 4 s, even if the distances are
different? Hint: compare with spin-diffusion.
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13
13
83
13
13
13
Ad Bax
*1956
a DQF-COSY.
84
13
C- and
15
N-NMR experiments at
13
slowly than 1H-spins under the influence of a PFG. We can thus use a PFG to
rephase the magnetisation of 13C and rephase it with a 4 times smaller PFG after
the magnetisation has been transferred to 1H. 1H magnetisation that does not talk
to
13
C will not rephase and therefore remain unobservable. Great way for
Many of the small-molecule NMR experiments with PFGs have been pioneered
by James Keeler. They have made 2D NMR much faster.
James Keeler
OBS: each time a PFG is applied, the lock must be switched off temporarily
(something that is written into the pulse program).
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Selective pulses
The excitation profile of a pulse is closely related to (though not identical with)
the Fourier transform of the pulse shape. The Fourier transform of a rectangle is
the sinc function (sin(x)/x). To deliver the maximal power in the shortest time,
hard pulses have a rectangular shape. The longer a rectangular pulse, the narrower
the central excitation band becomes, until it no longer covers the entire NMR
spectrum. For a very long pulse, it may cover only a single NMR resonance, but
there would be excitation sidebands as shown in the figure below (produced by
Max Keniry, RSC).
The trick with the Gaussian pulse shape is that it avoids excitation sidebands,
delivering more selectivity for the same pulse duration.
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The selective pulses can be used to perform 2D NMR experiments using a very
narrow spectral width in the indirect dimension, corresponding to a very large
increment of the evolution time and, hence, a short overall experimental time. It is
even quite possible to excite a single resolved resonance, reducing the 2D NMR
experiment to a 1D experiment. (See Kessler et al., Magn. Reson. Chem. 29, 527557 (1991).)
Selective pulses are also used to suppress intense solvent signals, without
affecting the rest of the NMR spectrum. (See Piotto et al., J. Biomol. NMR 2,
661-665 (1992).)
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Appendix
The Product Operator Formalism
In 1984, Richard Ernst published a formalism that made it possible for the first
time to predict the outcome of NMR pulse sequences in a simple way (Srensen,
Eich, Levitt, Bodenhausen, Ernst (1984) Product operator formalism for the
description of NMR pulse experiments. Progress NMR Spectr. 16, 163-192).
The article itself is a bit difficult to read, but it boils down to a few simple rules
summarized below.
By way of introduction, assume a 2-spin system with spins A and B. Start from
spin A. At equilibrium, we have longitudinal magnetisation along the z-axis, Az.
Following a 90ox pulse, we have magnetisation along the y axis, -Ay.
Note 1: Ay represents all multiplet components of spin A (in this case its just a
doublet), i.e. during free precession, the different multiplet components of -Ay
will fan out in the transverse plane.
Note 2: Immediately after the 90o pulse all multiplet components of A are still
aligned. This is in-phase magnetisation. If we were to apply a pulse on B, this
would not have any influence on the components of A.
In the product operator formalism, in-phase magnetisation is represented by Ax
and Ay.
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and denoted AyBz in the product operator formalism (i.e. spin A is transverse
along the y-axis and the orientation of the two double components depend on the
spin state of B in half the molecules itll be , in the other half ). If we apply,
e.g., a 180o pulse on B, this would invert the A magnetisation!
Free precession:
As spin A precesses, it converts from in-phase to antiphase magnetisation and
back again under the influence of the J-coupling:
-Ay -Ay cos(Jt) + AxBz sin(Jt)
AxBz AxBz cos(Jt) + Ay sin(Jt)
For t = 1/(2J), the conversion between in-phase and antiphase magnetisation is
complete.
On top of that, there is the influence of chemical shift evolution:
Ax Ax cos(t) + Ay sin(t)
90
Ay Ay cos(t) - Ax sin(t)
If the offset from the spectrometer frequency is zero ( = 0), there is no chemical
shift evolution. The way to remember the x and y and signs is to look at the
projections of a single vector onto the x- and y-axis:
Let the vector move anti-clockwise and it will change its direction: from x to y to
x to y to x etc. If the vector started on x, it will now be described by
x cos + y sin
i.e. the sum of the projections onto the x- and y-axes. If it started on y, it will be
described by
-y cos + x sin
and so on.
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Advanced Problem: Using the trigonometric relationships above, show that Ax creates
a spectrum with two components as expected for an in-phase doublet. Hint:
only in-phase magnetisation induces a current in the detection coil.
Advanced Problem: Show that AxBz creates a spectrum with a positive and a negative
doublet component, as expected for an antiphase doublet. Hint: describe the
evolution into in-phase magnetisation under the influence of chemical shift
and coupling evolution.
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Names:
Az: longitudinal magnetisation of spin A
Ax: in-phase x-magnetisation of spin A
Ay: in-phase y-magnetisation of spin A
AxBz: antiphase x-magnetisation of spin A, or more specifically, x-magnetisation of
spin A antiphase with respect to spin B
AyBz: antiphase y-magnetisation of spin A, or more specifically, y-magnetisation of
spin A antiphase with respect to spin B
AxBx, AyBy, AxBy and AyBx: two-spin coherence of spins A and B
AzBz: longitudinal two-spin order of spins A and B
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(unobservable z-magnetisation)
AxBz -AxBy
Ax Ax
AyBz AzBy
Hence, the diagonal peak and cross-peak are 90o out of phase, i.e. if the crosspeak is absorptive, the diagonal peak is dispersive (producing a bad baseline
because dispersive peaks are so broad). This is true in both dimensions, because
sin(At1)cos(Jt1) is an odd function (point symmetry with respect to zero),
whereas sin(At1)sin(Jt1) is an even function (mirror symmetry with respect to
zero).
In this way, the outcome of most NMR pulse sequences can be calculated.
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Note: the product operator formalism described above applies to spin systems
only. In the literature, terms containing 2 or more spins are multiplied by
normalisation factors (two-spin terms are multiplied by 2, three-spin terms by 4,
etc.) to maintain the magnitude of the product operators when A = , B= , etc.
Thus, when in-phase magnetisation interconverts with anti-phase magnetisation:
Ax 2AyBz, and 2AxBz Ay. These normalisation factors complicate the
writing without contributing to understanding.
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