NMR workshop (Part II)

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NMR workshop (Part II) USD, summer
2006 Available online at http//www.usd.edu/gsere
da Literature Timothy D. W. Claridge
High-Resolution NMR Techniques in Organic
Chemistry, 1999, Pergamon
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Outline 1. Summary of the RF-spin
interaction 2. Specifics of 13C
spectroscopy. Broad band decoupling 3.
Nuclear Overhauser effect 4. Different types
of 13C spectra. Decoupling and NOE 5. The
spin echo pulse sequence 6. The APT
experiment 7. Polarization transfer 8.
The DEPT experiment 9. 2D-NMR. Source of the
second dimension. COSY experiment 10. TOCSY
and 1D-TOCSY experiments 11. NOE and NOESY
experiments 12. Heteronuclear correlation
and indirect detection. HETCOR and HMQC
experiments 13. Solvent suppression 14.
Application of NMR in the student organic
chemistry laboratory
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1. Summary of the RF-spin interaction
A magnetic nucleus (a spin) rotates around the
magnetic field B0 with the Larmur frequency w0.
We want to affect the spin with the magnetic
component B1 of the radiofrequency (rf). The B1
is too weak to compete with B0. How to eliminate
the competition?
1. Apply very strong rf. (Not practical, not
selective) 2. Stop rotation of the spin around B0
(How to achieve this?) a. Remove the magnetic
field B0 (Not practical). b. Rotate with the spin
at the w0 frequency and apply B1 in the rotating
system (rotating frame) Consequence The field B1
must oscillate at the w0 frequency. Advantage
Selectivity toward spins, rotating at the
frequencies, close to w0.
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There are three possible situations 1. The rf
frequency is exactly wo. There is no competition
from B0. The spin is affected the most. That is
the resonance (NMR)
What happens during the resonance? a. The
oscillating rf pushes the rotating spin forward,
trying to accelerate it, but it is impossible,
because w0 is determined only by the magnetogyric
ratio of the spin and magnitude of the magnetic
field B0. The energy must drain somewhere and
promotes the spin to a higher energy state (spin
flip due to rotation around the magnetic field
B1). b. The phases of the spins are affected.
They adjust themselves to the phase of rf and
become correlated. 2. The rf frequency is far
from wo. The field B1 is not competitive with B0.
The spin is not affected by rf. 3. The rf
frequency is close to wo. The spin slowly rotates
with respect to the oscillating rf and changes
its phase. It allows us to play with the phases
and acquire more information about the system,
than just the set of resonance frequencies.
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Stationary frame, single spin
Rotating frame (w0), single spin
Problem A single spin does not emit rf
(otherwise it would eventually lose energy and
stop rotating, which is impossible) and,
consequently, is hard to be sensed and
monitored. Measuring absorbance of rf is hard
technically.
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Rotating frame (w0), many spins
Note If Mz is changed after the sampling pulse,
it does not affect the signal (too late).
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Different orientation of a spin in the magnetic
field may create different additional magnetic
fields at other spins and, consequently change
their Larmur (resonance) frequencies. If the
Larmur frequency of a spin of interest (A)
depends on the orientation of a neighboring spin
(B), it will resonate at slightly different
frequencies (n1 and n2), when the spin B is in
the spin state a or in the spin state b. In the
frame of reference, rotating at n0 (center of the
doublet), the components n1 and n2 will rotate
(evolve) with the same speed in oposite
directions.
Doublet (if there is one neighbor B)
A resonates at n1
B is in the spin state a
A resonates at n2
B is in the spin state b
Triplet (if
there are two neighbors B) A
resonates at n1
B1 is in the spin state a

B2 is in the spin state a
A resonates at n2
B1 is in the spin state a or b

B2 is in the spin state b
or a A resonates at n3
B1 is in the spin state b

B2 is in the spin state b
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2. Specifics of 13C spectroscopy. Broad band
decoupling

• Low natural abundance of 13C (less than 1)
• Consequence presence of more than one 13C per
  molecule is
• very unlikely, so no 13C-13C coupling is normally
  observed
• b. Low magnetogyric ratio for 13C, which makes
  the nucleus less
• responsive to the magnetic field
• Consequence the resonance occurs at a lower
  frequency, than for 1H.
• (13C would resonate at 50 MHz, if 1H resonates at
  200 MHz in the same magnetic field). It allows us
  to observe 1H and 13C spectra separately.
• c. Larger relaxation times (gt 5 sec) than for 1H
  (less than 5 sec).
• Consequence Integration of signals requires
  larger relaxation delays,
• which need to be optimized before the experiment
• Consequence from a, b, and c the experiment
  takes longer (normally 256 scans about 4 sec each
  vs 8 scans about 2 sec each for 1H) and requires
  higher concentration of the material.

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Is it possible to suppress all couplings at once
and simplify the spectrum? Decoupling with a
particular nucleus (intense irradiation at its
resonance frequency during acquisition) rapidly
interchanges its energy levels, averages
frequencies of the components of the multiplet,
which causes the loss of splitting in the
multiplet. Another effect of coupling is
saturation loss of phase coherence while a- and
b-populations are equal. Therefore, a saturated
resonance produces no signal. Depending on the
duration of the decoupling pulse, decopling can
be selective (saturation of one particular
resonance) or broad band (saturation of all
resonances of a particular nuclide, for instance,
1H).
Excitation profiles of two rf pulses of different
duration
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Homonuclear decoupling suppression of coupling
between the same type of nuclides (for instance
1H - 1H). Heteronuclear decoupling suppression
of coupling between different types of nuclides
(for instance 1H 13C). Total (broad band)
homonuclear decoupling is impossible without loss
of the whole spectrum. While taking a 13C
spectrum we do not care about 1H signals.
Consequence We can suppress 13C 1H couplings
all at once. It is called broad band decoupling
and performed by making the decoupler to cover
the whole 1H frequencies range.
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3. Nuclear Overhauser effect

• Suppose, we have two coupled nuclei I and S and
  want to decouple
• them by irradiating the system at the resonance
  frequency of S.
• It results in
• Disappearance of the signal S
• b) Suppression of the coupling between I and S.
• c) Change in the intensity of the signal I (side
  effect of decoupling).
• The effect c) is called the Nuclear Overhauser
  effect or NOE.
• If the intensity I increases, NOE is said to be
  positive.
• If the intensity I decreases, NOE is said to be
  negative.
• The source of NOE correlated relaxation of both
  nuclei I and S,
• so that the total spin of the system changes by 1
  (transition W2, both I and S increase or decrease
  their spins when the molecules are tumbling at
  the frequency of I plus the frequency of S
  characteristic for small, faster molecules),
• or does not change at all (transition W0, either
  I or S increases its spins, and another nucleus
  decreases its spin when the molecules are
  tumbling at the frequency of I minus the
  frequency of S characteristic for large, slower
  molecules).

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Consequences of the relaxation transitions W2 and
W0
1. a) to b) Populations of the ground and
excited states of S become equal. The signal S
disappears. 2. b) to c) The W2 relaxation
increases the population of a ground state of I
and decreases population of its excited
state. The positive NOE develops (characteristic
for small molecules). 3. b) to d) The W0
relaxation increases the population of an excited
state of I and decreases population of its ground
state. The negative NOE develops (characteristic
for large molecules).
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4. Different types of 13C spectra. Decoupling and
NOE
Two effects of decoupling (decoupling itself and
NOE) can be separated, resulting in different
types of 13C spectra.
1. Gated decoupling
The NOE develops before and during the
acquisition. The coupling is retained, because
the nucleus S does not jump back and forth during
the acquisition, so the nucleus I precesses at
two frequencies, depending on the spin of S. It
produces a coupled spectrum with NOE.
2. Inverse-gated decoupling
The NOE develops and affects the longitudinal
magnetization, but since acquisition is already
in progress, the registered transversal
magnetization is not affected, so the NOE does
not show up in the spectrum. The coupling is
suppressed, because the nucleus S jumps back and
forth during the acquisition. The nucleus I
precesses at two frequencies, depending on the
spin of S, but it is all averaged in the NMR time
scale. It produces a decoupled spectrum without
NOE.
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3. Power-gated decoupling
The decoupler is on throughout the experiment,
so both decoupling and NOE take place. During
the lengthy relaxation delay, the decoupler
power is lower to avoid overheating the sample
and to increase the lifetime of the probe. It
produces a decoupled spectrum with NOE (the most
popular routine 13C spectrum).
4. No decoupling
It produces a coupled 13C spectrum without NOE.
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Different types of 13C spectra of isobutyl
butyrate
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Influence of relaxation delay on the integration
of 13C signals
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5. The spin echo pulse sequence
With respect to the rotating frame, spins with
higher Larmor frequencies rotate forward, and the
spins with lower Larmor frequencies rotate
backwards. If we let them rotate for a certain
time t and flip them by 180o about the Y axis,
they will refocus after another period t.
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Spins (nucleus A), which are coupled with another
spin (X), or in other words, are components of a
multiplet, behave differently, if the spin X also
undergoes a 180o pulse (spin flip). Each
component of a multiplet may become coupled with
another spin state of X, change its Larmor
frequency and drift the opposite way. It causes
defocusing of components of multiplets, rather
than refocusing.
That is how we can sense if the spin is coupled
with another spin.
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6. The APT experiment
Evolution of some multiplet components over the
time (differences in 13C chemical shifts are
ignored)
The components of multiplets drift at different
speeds and end up at opposite phases after the
time 1/J. Problem It works only for carbons
with same chemical shifts.
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SEFT (Spin-Echo Fourier Transform) sequence
1. The decoupler-gated variant Purpose to
refocus chemical shifts, but still distinguish
different multiplets (see the previous slide).
During the first 1/J period we let the
multiplets arrive to opposite phases. During
the second 1/J period we stop their evolution by
decoupling and refocus carbons with different
chemical shift by applying a 180o pulse (spin
echo sequence)
2. The pulsed variant At the end of the first
1/2J period we apply a 180o pulse on the
X-channel to refocus carbons with different
chemical shifts. (spin echo sequence) At the
same time we apply a 180o pulse on the 1H-channel
to keep components of carbon multiplets from
refocusing and allow them to arrive to opposite
phases at the end of the second 1/2J period. It
results in the same situation as on the previous
slide, but with opposite phases.
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APT (Attached Proton Test) is the decoupler-gated
variant of The SEFT sequence, modified in two
ways 1. The first pulse is less than 90o to
optimize acquisition, but it leaves z
components. 2. One more 180o pulse inverts back
these undesired components to z.
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7. Polarization transfer
Polarization transfer enhances signal intensity
by transferring the greater population
differences of high-g spins onto their coupled
low-g partners, leading to a signal enhancement
by a factor of ghigh/glow (For the 1H/13C pair
by the factor of about 4). It can be achieved by
selective inversion of population of a high-g
component of a multiplet.
Before the inversion
After the inversion
Problem How to simultaneously and selectively
invert populations of one half of each
miltiplet?
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8. The DEPT experiment
The refocused INEPT (Insensitive Nuclei Enhanced
by Polarization Transfer) sequence Purpose
generate a greater population difference for the
less sensitive nucleus along the z-axis.
On the first step, polarization is transferred to
CH-carbons (doublets). The spin-echo sequence
ensures that the process is independent on
proton chemical shifts. Then a 90o pulse samples
the enhanced population difference of 13C and
allows components of all CH-doublets to refocus.
The spin-echo sequence ensures that the process
is independent on 13C chemical shifts.
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The farthest signals of the CH3 quadruplet also
end up in antiphase, so only CH-doublets show up
in the spectrum. To detect other multiplets,
durations of both steps should be changed.
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The DEPT (Distortion Enhancement by Polarization
Transfer) Sequence makes the same effect as the
refocused INEPT sequence, but simplifies editing
of the resulting spectrum. 1. The 90o pulse on
the X channel starts together with the 180o pulse
on the 1H channel and triggers coherent evolution
of both 1H and X spins when the components of the
multiplet are antiphase. 2.The q-pulse on the
1H-chanel transfers polarization to the
transverse plane on the X-channel. 3. Instead
of varying the spin-echoes durations, the width
of the third pulse on the proton channel is
adjusted to each separately recorded
multiplet. 4. Two 180o pulses serve in two
spin-echo sequences, eliminating the influence of
1H and X chemical shifts.
Due to the polarization transfer, the signal to
noise ratio for DEPT-spectra are better than for
APT-experiments
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9. 2D-NMR. Source of the second
dimension. COSY
experiment
To add a second dimension to the frequency
domain, we need to add a variable to the time
domain. It is accomplished by running a series of
experiments with a variable time somewhere in the
pulse sequence.
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The COSY (Correlation Spectroscopy) sequence
It is a homonuclear analogue of the basic INEPT
sequence. A part of magnetization not
transferred to the coupling partner, precesses
with the original resonance frequency n1 during
both t1 and t2 periods. This results in a
diagonal peak in the frequency domain (n1-n1). A
part of magnetization, transferred to the
coupling partner, precesses with the original
resonance frequency n1 during t1 and with the
partners resonance frequency n2 during t2. This
results in an off-diagonal peak in the frequency
domain (n1-n2).
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10. TOCSY and 1D-TOCSY experiments
Spin-echo sequence
Spin-lock sequence
Spin-lock sequence is the Spin-echo sequence,
applied continuously. The simplest spin-lock
sequence is just a continuous pulse.
The Spin-lock sequence makes all spins strongly
coupled (differences in chemical shifts are less
than coupling constants)
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TOCSY (Total Correlation Spectroscopy) sequence
In contrast to COSY, the TOCSY sequence has a
Spin-lock pulse, instead of a 90o pulse. All
coupled spins (each spin and its partner) undergo
a 180o pulse, so all multiplet components are
not locked and continue to evolve, propagating
further along the chain during the mixing time tm
(tens of milliseconds). All spins become
strongly coupled and lose their identity. It
leads to sharing of coherence between all spins
and produces additional (comparing with COSY)
off-diagonal peaks between signals, not coupled
directly, but belonging to the same spin system
in the molecule.
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1D-TOCSY sequence
This sequence is close to TOCSY sequence, but
instead of a 90o pulse, a particular spin is
selectively excited by a field gradient or by one
of numerous sequences for selective excitation.
Additionally, there is no need to array the time
between excitation and the spin-lock. This
experiment allows to see how coupling propagates
along the chain of spins until it embraces the
whole spin-system. It allows observation of
spin-systems (fragments) of a molecule
separately. Examples 1. Acidic and
alcoholic part of esters 2. A molecule in
the presence of impurities 3. Fragments of
individual amino-acids in a molecule of peptide
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11. NOE and NOESY experiments
The NOE effect, which is observed when saturation
of 1H-resonances changes intensity of coupled 13C
signals, is not limited to coupled spins. NOE
may be observed between non-coupled spins,
located within 5A from one another. Hence, it
provides significant stereochemical information.
For small molecules NOE is usually positive, for
large molecules negative. For some molecules
it is close to zero. So, NOE results may tell us
yes or may be, but never no.
NOE-difference sequence
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The steady-state NOE is measured by continuous
saturation of the spin of interest. The transient
NOE is initiated by a one-moment population
disturbance and measured by a 2D-experiment,
called NOESY (Nuclear Overhauser Effect
Spectroscopy).
The NOESY sequence is similar to COSY sequence,
but after the second 90o pulse, instead of
acquisition we wait for the mixing time tm to let
the NOE develop. The third 90o pulse samples the
NOESY affected population by placing the NOESY
component of magnetisation to the transverse
plane.
The NOESY component accumulates along the Z-axis,
which makes it insensitive to the rf phase.
Therefore, the COSY components can be removed by
phase cycling.
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12. Heteronuclear correlation and indirect
detection. HETCOR and HMQC experiments
The HETCOR (Heteronuclear Correlation) sequence
The refocused INEPT sequence (a reminder)
The INEPT sequence transfers polarization from 1H
to 13C and acquires information on which
hydrogens and which carbon are connected. To
plot this information as a spectrum, we just need
to add the second dimension (left of the dotted
line) to the refocused INEPT sequence (right of
the dotted line) and a 180o pulse to refocus the
couplings of X (effective decoupling during t1).
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The HMQC (Heteronuclear Multiquantum Coherence)
sequence
The first 90o pulse excites protons and their
magnetization is transferred to carbons by the
90o pulse on the carbon channel. Then, during the
variable time t1, proton and 13C spins evolve
coherently and sampled by the last 90o pulse on
the carbon channel. To remove the influence of
proton chemical shifts during t1, a spin-echo
sequence is applied to the proton channel. The
acquisition starts after another period 1/2J to
allow components of proton multiplets refocus
(spin-echo sequence, applied to the whole time
range). The extent of coherent evolution depends
on the time t1, which brings correllation between
proton and 13C frequencies to the FID.
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13. Solvent suppression
Before the acquisition the solvent resonance is
saturated for the period d2 (decoupler mode
nyn). The standard delay time d1 is not needed
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Protons chemically exchangeable with the solvent,
are also suppressed.
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14. Application of NMR in the student organic
chemistry laboratory
Dehydration of 4-methylpentanol-2 (DEPT spectrum)
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Selectivity of oxime formation
The reaction is very selective
A pseudo-singlet in the aromatic area
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Identification of unknown alcohols and acids by
esterification
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Iodochlorination of styrene
An example of the AB system
Practicing the concept of diastereotopic groups
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Synthesis of a porphyrin. Practicing the concept
of aromaticity and setup of the tof parameter
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Explicit identification of two singlets versus
one doublet, using COSY