NMR Spectroscopy

Report
2-D NMR
Spectroscopy
6-1
PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D NMR Basics.
• In actuality, the techniques we have already covered 1H, 13C, and DEPT are 2D (frequency vs. intensity) however, by tradition the intensity component is
dropped when discussing dimensionality
• In 2-D techniques, many FIDs (proto-NMR spectra) are taken one after
another, with some acquisition variable or pulse sequenced varied by small
increments
• Since each FID is a collection of digitized data points in the first dimension
(say 10 points to make a spectrum) if 10 spectra are accumulated with an
incremental change in variable, an FT can be performed in the other
dimension
1-D FID
1-D spectra, each
with an incremental
variable change
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CHEM 430 – NMR Spectroscopy
FTs can be performed on
the vertical data sets
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2-D NMR
Spectroscopy
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D NMR Basics.
• The first perturbation of the system (pulse) is called the preparation
of the spin system.
• The effects of this pulse are allowed to coalesce; this is known as the
evolution time, t1 (NOT T1 – the relaxation time)
• During this time, a mixing event, in which information from one part
of the spin system is relayed to other parts, occurs
• Finally, an acquisition period (t2) as with all 1-D experiments.
Preparation
Evolution
Mixing
t1
CHEM 430 – NMR Spectroscopy
Acquisition
t2
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2-D NMR
Spectroscopy
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.
• H-H COrrelation SpectroscopY (COSY):
• The pulse sequence for COSY is as follows:
90x
90x
t2
t1
•
A 90o pulse in the x-direction is what we used for 1-D 1H NMR
•
Here, after a variable “mixing” period, a second 90o pulse is performed,
followed by acquisition of a spectrum
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2-D NMR
Spectroscopy
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.
• Consider a simple spectrum with one resonance (CHCl3):
B0
z
M
y
x
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2-D NMR
Spectroscopy
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.
• We pulse the sample with our standard 90ox tilting magnetization into y
B0
z
90x
M
y
x
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2-D NMR
Spectroscopy
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.
• After a short time t the vector begins to evolve around the z-axis
B0
z
90x
y
M
x
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2-D NMR
Spectroscopy
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.
• This vector now has an x-component and y-component
B0
z
90x
t1
Mcoswt1
y
Msinwt1
M
x
CHEM 430 – NMR Spectroscopy
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2-D NMR
Spectroscopy
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.
• If a 90ox pulse is applied again only the y component is rotated to -z
B0
z
90x
90x
t1
y
Msinwt1
Mcoswt1
x
M
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2-D NMR
Spectroscopy
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.
• If we now detect in the xy plane, only the x-component remains
B0
z
90x
90x
t2
t1
Signal:
Msinwt1
x
y
Mx
7.0
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2-D NMR
Spectroscopy
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.
• Repeat the experiment, but let time evolve by a longer increment
B0
z
90x
y
M
x
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2-D NMR
Spectroscopy
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.
• This vector now has a greater x-component than before
B0
z
90x
t1
Mcoswt1
Msinwt1
x
y
M
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2-D NMR
Spectroscopy
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.
• If a 90ox pulse is applied again only the y component is rotated to -z
B0
z
90x
90x
t1
y
Msinwt1
Mcoswt1
x
M
CHEM 430 – NMR Spectroscopy
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2-D NMR
Spectroscopy
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.
• Now we will detect a larger x-component than before
B0
z
90x
90x
t2
t1
Signal:
Msinwt1
x
y
Mx
7.0
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2-D NMR
Spectroscopy
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.
• In the COSY experiment a new ‘spectrum’ is acquired at increments of t1
• If we stack the array of spectra evolving at t1 increments, notice how we now
have a new FID of sorts in the orthogonal coordinate!
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2-D NMR
Spectroscopy
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.
• If we carry out a Fourier Transform in this other coordinate, we generate a 2D spectrum where the CHCl3 peak shows up at 7.27 on both axes.
• The peak is more a sharp cone in shape
FT
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2-D NMR
Spectroscopy
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.
• Now let’s make this more complex; but what occurs now is a simplification!
B0
z
M
y
x
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2-D NMR
Spectroscopy
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.
• Let’s pulse a sample with two nuclei that are spin coupled.
B0
z
90x
M
y
x
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2-D NMR
Spectroscopy
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.
• By T2 relaxation vector begins to diverges as +J/2 and –J/2
B0
z
B0
z
y
y
M
x
M
x
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2-D NMR
Spectroscopy
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.
• The two nuclei are precessing at different w and decaying at different T1 BUT
they share a frequency of oscillation of resultant x-component about the zaxis!!!
z
z
y
y
M
x
M
x
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2-D NMR
Spectroscopy
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.
• So in the 1st dimension, the spectra appear “normal”, but in the second
dimension these two nuclei share a oscillation frequency of x-component
• If we stack the spectra we see that they show an artifact of related spin at the
frequency of their coupling partner
• Rather than view the 3-D map, it is customary to interperet the 2-D
spectrum as viewing from overhead
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2-D NMR
Spectroscopy
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
2D COSY.
• Also keep in mind that J values have a sign and the vectors they generate
have a – and + component, so in reality:
f1
f2
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
How to use 2D COSY.
• Consider the COSY spectrum
of butyl propanoate
• Remember all resonances
share their own variation of
x-component in the COSY
experiment.
• The peaks along the diagonal
in the COSY spectrum show
this relationship and can be
ignored.
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
How to use 2D COSY.
• The ‘normal’ 1-D 1H
spectrum is placed on the F1
and F2 axes as a reference.
• This spectrum is obtained
separately!
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
How to use 2D COSY.
• To find a pair of coupling
partners simply find the
cross peak relationships
• For example the resonance at
d 4.05 we can identify by
chemical shift as being
adjacent to the sp3 oxygen of
the ester.
• We find it is coupled to the
resonance at d 1.65
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
How to use 2D COSY.
• We now use the d 1.65
resonance to find the next
coupling partner
• We see it is coupled to the
adjacent resonance at d 1.4
• Likewise the d 1.4 resonance
is coupled to the resonance
at d 0.95
• This resonance only has the d
1.4 as a coupling partner, so
the chain ends.
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
How to use 2D COSY.
• Likewise we can deduce the
ethyl chain attached to C=O
as a separate coupled family
• The only drawback of the
COSY experiment is it cannot
‘see through’ parts of the
molecule that have no 1H-1H
coupling (2JHH or 3JHH)
• These include 4o carbons,
C=O, 3o amines, -O-, -S-, etc.
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2-D NMR
Spectroscopy
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
DQFCOSY.
• Double quantum filtered - an extra
pulse is added after the second
COSY pulse, and phase cycling
converts multiple quantum
coherences into observable
magnetizations.
• The resulting 2D spectrum lacks all
singlets along the diagonal
• The experiment has surplanted the
COSY-45 in the text
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2-D NMR
Spectroscopy
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PROTON-PROTON CORRELATION THROUGH J-COUPLING
DQFCOSY.
• An important feature of the
experiment is that double quantum
filtration allows both diagonal and
cross peaks to be tuned into pure
absorption at the same time.
• This reduces the size of all diagonal
signals and permits cross peaks
close to the diagonal to be
observed.
• The only disadvantage of DQF–
COSY is a reduction in sensitivity
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2-D NMR
Spectroscopy
6-2
PROTON-HETERONUCLEUS CORRELATION
HETCOR.
• The 13C-1H COSY (HETeronuclear CORrelation) experiment correlates 13C
with directly attached 1H via 1JCH couplings.
• Since the frequency domains F1 and F2 are for different nuclei we do not
observe diagonal peaks as in 1H-1H COSY
• During the evolution time the large 1JCH is used for polarization transfer, so
only 13C directly bound to 1H are detected
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2-D NMR
Spectroscopy
6-2
PROTON-HETERONUCLEUS CORRELATION
HETCOR.
• Note how we can now determine that two protons are on the same carbon:
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2-D NMR
Spectroscopy
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PROTON-HETERONUCLEUS CORRELATION
HETCOR.
• The principle disadvantage of HETCOR is total acquisition time. Typically 58 times the length of the corresponding 13C experiment.
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2-D NMR
Spectroscopy
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PROTON-HETERONUCLEUS CORRELATION
HSQC/HMQC.
• One way around the sensitivity problem with HETCOR is to observe the 1H
in the 13C-1H system and study 1JHC rather than the non-abundant 13C.
• The 2D HSQC (Heteronuclear Single-Quantum Correlation) experiment
permits to obtain a 2D heteronuclear chemical shift correlation map
between directly-bonded 1H and X-heteronuclei (commonly, 13C and 15N).
• One interesting artifact of the HSQC experiment is the ability to create a lowresolution 1-D 13C spectrum from the 2-D data!
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2-D NMR
Spectroscopy
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PROTON-HETERONUCLEUS CORRELATION
HSQC/HMQC.
• The data is interpreted like HETCOR; the 13C spectrum on the F1 axis is
acquired separately and actually takes longer than the 2D acquisition!
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2-D NMR
Spectroscopy
6-2
PROTON-HETERONUCLEUS CORRELATION
HMBC.
• The 2D HMBC experiment permits to obtain a 2D heteronuclear chemical
shift correlation map between long-range coupled 1H and heteronuclei
(commonly, 13C).
• It is widely used because it is based on 1H-detection, offering high sensitivity
when compared with the 13C-detected.
• In addition, long-range proton-carbon coupling constants can be measured
from the resulting spectra.
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2-D NMR
Spectroscopy
6-2
PROTON-HETERONUCLEUS CORRELATION
HMBC.
• The power of this technique is it allows us to “see” through a quarternary
carbon center!
• The drawback is that for small molecules with tight ring or fused-ring
systems (like many of the unknowns) everything may be correlated to
everything else!
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2-D NMR
Spectroscopy
6-2
PROTON-HETERONUCLEUS CORRELATION
HMBC.
• In this example we see ipsenol and
each of the ‘5-C domains’ that make
up the molecule.
• For example C-4 is within 3JHC of H3, H-3’, H-2, OH, H-5 and H-5’
• C-7 can be seen to be within 3
bonds of 5/5’ through the 4o center
at C-6!
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2-D NMR
Spectroscopy
6-P
PROBLEMS - 1
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Spectroscopy
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Spectroscopy
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Spectroscopy
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Spectroscopy
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Spectroscopy
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2-D NMR
Spectroscopy
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2-D NMR
Spectroscopy
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PROBLEMS – 2 - HSQC
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2-D NMR
Spectroscopy
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PROBLEMS - 3
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2-D NMR
Spectroscopy
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2-D NMR
Spectroscopy
6-2
PROTON-HETERONUCLEUS CORRELATION
TOCSY.
• TOtal Correlation SpectroscopY ( TOCSY). By spin locking the protons
during the second COSY pulse, the chemical shifts of all the protons may be
brought essentially into equivalence.
• The initial pulse and the period occur as usual, but the second pulse locks
the magnetization along the y-axis so that all protons have the spin lock
frequency.
• All coupled spins within a spin system then become closely coupled to each
other, and magnetization is transferred from one spin to all the other
members, even in the absence of J couplings.
CHEM 430 – NMR Spectroscopy
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PROTON-HETERONUCLEUS CORRELATION
NOESY.
• The spectrum on the right shows the TOCSY
experiment for lysine.
• TOCSY has particular advantages for large
molecules, including enhanced sensitivity
and, if desired, the phasing of both diagonal
and cross peaks to the absorption mode.
• The process of identifying resonances within
specific amino acid or nucleotide residues is
considerably simplified by this procedure.
Each residue can be expected to exhibit cross
peaks among all its protons and none with
protons of other residues.
CHEM 430 – NMR Spectroscopy
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2-D NMR
Spectroscopy
6-2
PROTON-HETERONUCLEUS CORRELATION
NOESY.
• After the second pulse in COSY the NOE mechanism can modulate the cosine
component of the magnetization along the z axis.
• The frequency of modulation is the frequency of the magnetization transfer
partner from dipolar relaxation
• After a suitable fixed period (tm, the mixing period), during which this
modulation is optimized, the cosine component may be moved to the xy
plane by a third pulse and may be detected along the y axis during a
acquisition period.
CHEM 430 – NMR Spectroscopy
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2-D NMR
Spectroscopy
6-2
PROTON-HETERONUCLEUS CORRELATION
NOESY.
• Because the frequency of magnetization of some nuclei during moves to
another value during t1 and is observed at the new frequency during tm , the
2D representation of this experiment exhibits cross peaks.
• When the cross peaks derive from magnetization transfer through dipolar
relaxation, the 2D experiment is called NOESY (NOE SpectroscopY).
• The duration of the fixed time tm depends on T1 and the rate of NOE buildup.
• In the NOESY experiment valuable information can be ascertained about the
distance between various protons within a molecule (< 5Å).
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PROTON-HETERONUCLEUS CORRELATION
NOESY.
COSY
NOESY
CHEM 430 – NMR Spectroscopy
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PROTON-HETERONUCLEUS CORRELATION
NOESY.
Overlapped COSY and NOESY
NOESY in faded color
CHEM 430 – NMR Spectroscopy
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2-D NMR
Spectroscopy
6-2
PROTON-HETERONUCLEUS CORRELATION
NOESY.
At least three factors complicate the analysis of NOESY spectra.
1. COSY signals may be present from scalar couplings and may interfere
with interpretations intended to be based entirely on interproton
distances.
2. In small molecules, the NOE builds up slowly and attains a theoretical
maximum of only 50% — Because a single proton may be relaxed by
several neighboring protons, the maximum is much less than 50%.
3. In addition to its transfer directly from one proton to an adjacent
proton, magnetization may be transferred by spin diffusion. In this
mechanism, magnetization is transferred through the NOE from one
spin to a nearby 2nd spin and then from the 2nd to a 3rd spin that is close
to the 2nd spin, but not necessarily to the first one. These multistep
transfers can produce NOESY cross peaks between protons that are not
close together.
CHEM 430 – NMR Spectroscopy
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2-D NMR
Spectroscopy
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PROTON-HETERONUCLEUS CORRELATION
ROESY.
• The Rotating frame Overhauser Effect SpectroscopY utlizes spin-locking
(like TOCSY) to ameliorate the drawbacks of NOESY for small molecules.
• The pulse sequence is as follows –data interpretation is the same as NOESY
CHEM 430 – NMR Spectroscopy
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2-D NMR
Spectroscopy
EXAMPLES
6-E
VGSE: Valine-Glycine-Serine-Glutamic acid.
CHEM 430 – NMR Spectroscopy
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2-D NMR
Spectroscopy
EXAMPLES
6-E
VGSE: Valine-Glycine-Serine-Glutamic acid.
CHEM 430 – NMR Spectroscopy
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2-D NMR
Spectroscopy
EXAMPLES
6-E
VGSE: Valine-Glycine-Serine-Glutamic acid.
CHEM 430 – NMR Spectroscopy
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2-D NMR
Spectroscopy
EXAMPLES
6-E
VGSE: Valine-Glycine-Serine-Glutamic acid.
CHEM 430 – NMR Spectroscopy
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2-D NMR
Spectroscopy
EXAMPLES
6-E
VGSE: Valine-Glycine-Serine-Glutamic acid.
CHEM 430 – NMR Spectroscopy
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2-D NMR
Spectroscopy
EXAMPLES
6-E
VGSE: Valine-Glycine-Serine-Glutamic acid.
CHEM 430 – NMR Spectroscopy
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2-D NMR
Spectroscopy
EXAMPLES
6-E
VGSE: Valine-Glycine-Serine-Glutamic acid.
CHEM 430 – NMR Spectroscopy
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