CH437 CLASS 17
NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY 8
Synopsis. Two-dimension NMR correlation spectroscopy: COSY, NOESY, HETCOR, HMBC and
HMQC.
Two-Dimensional NMR Spectroscopy
In all the examples of NMR spectra that have been seen so far, the signal is
presented as a function of a single parameter, usually the chemical shift. Hence,
these are one-dimensional spectra. Complex molecules, such as proteins and
nucleic acids, are difficult to analyze fully by one-dimensional NMR because of
extensive overlap of signals. Multidimensional NMR techniques have been
developed to cope with such complex examples: in particular, H-H correlation
spectroscopy, better known as Correlation SpectroscopY (COSY) and H-non-H
correlation spectroscopy, known as HETeronuclear chemical shift CORrelation or
HETCOR. A COSY spectrum is usually a plot of 1H chemical shift on both the x
and y axes, whereas a HETCOR spectrum is usually a plot of 1H chemical shift
on one axis against the chemical shift of some other nucleus (usually
13C)
on the
other axis. For COSY and NOESY, the peaks in the whole spectrum (plotted as
contour lines derived from a stack plot – see later) lie along a diagonal line with
cross peaks, representing coupled nuclei.
Other kinds of correlation 2D NMR spectroscopy techniques include EXchange
SpectroscopY (EXSY), Heteronuclear Multiple Bond Coherence (HMBC),
Heteronuclear Multiple Quantum Coherence
(HMQC), J-spectroscopy and
Nuclear Overhauser Effect SpectroscopY(NOESY).
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The Basic COSY-90 Experiment
The basic COSY (strictly the COSY-90 or COSY-/2, since other versions are
used) pulse sequence is shown below:
Here,  is an incremental delay. Considering a single line in a complex multiplet,
a pulse of /2 causes the magnetization vector to precess in the xy plane at a
frequency 1 that depends on the its chemical shift and on each of the couplings
with which the multiplet is involved:
If the evolution time,  = 1/1, then at the end of that time, the vector will be
aligned along y and so the second /2 pulse will rotate the vector down along the
–z axis (see below). This results in inversion of the population distribution for that
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pair of energy levels and for all the connected transitions in the other resonances
to which the proton is coupled.
On the other hand, if is  = 1/21, then after that time, the vector will be aligned
along –y and the second /2 pulse will return the vector to the z axis, resulting in
normal population distribution:
Intermediate values of give intermediate results and repeating many
experiments with incremented  results in a set of spectra that have normal
chemical shift values and normal coupling, but each peak contains coded
information about the frequencies of all other peaks to which it is coupled. The
second Fourier transformation then gives a spectrum with 1H chemical shifts in
each dimension. The normal spectrum is now found along the diagonal because
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the intensity of each transition oscillates at 1 during t1 and because its detected
frequency in t2 is also 1. The off-diagonal “cross peaks” determine which
chemical shifts are connected to which, because the intensity of the 1 transition
is modulated during t1 by all the transitions to which it is connected.
Typical COSY contour plots are derived from stack plots that result from the
second Fourier transformation, as shown for ethyl vinyl ether below:
To analyze a COSY spectrum, firstly a diagonal is drawn through the
contour lines. The contour lines that are not on the diagonal (called “cross
peaks” or “off-diagonal peaks”) contain the coupling information.
For example, for ethyl vinyl ketone (above), draw a line from the first cross peak
A parallel to the y axis back to the diagonal. This gives the peaks at ca. 1.1 ppm
in the spectrum that corresponds to Ha. Now draw a line from A to the diagonal
parallel to the x axis. This gives the peaks at ca. 3.8 ppm, corresponding to Hb.
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The same thing can be done for cross peaks B and C: this will show Hc, Hd and
He are coupled. The following inceasingly complex examples serve to illustrate
the interpretation of COSY contour spectra.
For m-dinitrobenzene (above), the cross peaks indicate that H4 is coupled to H5,
H6 is coupled to H5 and H5 is coupled to both H4 and H6. Much weaker cross
peaks indicate long range coupling (small J) between H2 and H4, H6.
In the COSY spectrum of isopentyl acetate (below), it can be seen that the
protons of the two equivalent methyl groups (1) correlate with the methine proton
(2). Also evident is the correlation between the two methylene groups (3) and (4)
and between the methine proton (2) and the neighboring methylene protons (3).
The acetate methyl group (5) shows no cross peaks, because its protons are
“isolated” (they are not coupled to other protons).
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Citronellol gives a more complex example of the application of COSY NMR. The
1H
NMR spectrum shows considerable overlap of signals (e.g. between 2, 3, 4
and also between 2, 3, 8 and 9 proton signals). This example serves to illustrate
how COSY NMR can be used to identify certain important coupling interactions.
Firstly, the C5 methylene protons (~2 ppm) are clearly coupled to the alkene
proton, H6 (~5.2 ppm). Closer inspection reveals that H6 is coupled to the protons
of the two methyl groups (C8 and C9): this is an example of long-range allylic
coupling. Also, the methylene protons on C1 are coupled to two nonequivalent
protons on C2 (at ~1.4 ppm and ~1.6 ppm). They are nonequivalent because of
the adjacent chiral center (at C3). The coupling of the methyl protons on C10 with
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the methine proton H3 can also be seen, even though the H3 contour on the
diagonal line is obscured by overlaps.
NOESY Spectra
Here, the Nuclear Overhauser effect (NOE) is used to establish throughspace correlations between nuclei, usually between proton and proton.
Hence the plot resembles a complex COSY-90 plot, with the cross peaks
connecting resonances from nuclear spins that are spatially close. The NOESY
spectrum of codeine is shown below.
The interesting information is contained in the "cross-peaks", which appear at the
coordinates of 2 protons that have an NOE correlation.
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For example, the 3 cross-peaks indicated in squares on the plot above
distinguish between the 3 CH2 pairs: 5-18’; 16-17; 18-13.
The Basic HETCOR experiment
Two-dimensional NMR spectra that reveal heteronuclear shift correlations (like
13C-1H)
are
known
as HETCOR spectra
(HETCOR = HETeronuclear
CORrelation). The pulse sequence for HETCOR resembles that for COSY, but
simultaneous /2 pulses are applied to both 1H and
transfer magnetization from 1H to
13C.
13C
Now, since the
nuclei. These pulses
13C
magnetization was
encoded with 1H precession frequencies during  (the evolution time), the
13C
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signals that are detected during the acquisition time are modulated by the
chemical shifts of the coupled protons. Thus each
more peaks appearing on the f2 axis (the
13C
13C
nucleus may have one or
chemical shift axis) that correspond
to its chemical shift. The 1H chemical shift modulation causes the proton signal to
appear on the f1 axis at a value that not only corresponds to its own chemical
shift, but is also correlated with the chemical shift value of the
13C
nucleus to
which it is coupled.
In other words, the cross peaks in a HETCOR spectrum can be used to
identify which hydrogens are attached to which carbons (and vice versa).
The HETCOR spectra of 2-methyl-3-pentanone, isopentyl acetate and 4-methyl2-pentanol are shown below as examples.
Cross peak A indicates that the hydrogen atoms that are responsible for the
signal at ~0.9 ppm in the 1H spectrum (x axis) are attached to the carbon atom
responsible for the peak at ~6 ppm in the
13C
spectrum (y axis). Similarly, cross
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peak B (1H: ~1.0 ppm,
13C:
~18.7 ppm) and C (1H: ~2.5 ppm,
13C:
34.0 ppm) can
be used to assign carbon-hydrogen connectivities for the rest of the molecule.
Unprotonated
13C
nuclei do not appear in the HETCOR spectrum (in the above
example, the carbonyl carbon).
In the HETCOR spectrum of isopentyl acetate (above), the carbon peak at ~23
ppm and the proton peak at ~0.9 ppm correspond to the equivalent methyl
groups (1). The carbon peak at ~25 ppm and the proton multiplet at ~1.7 ppm
correspond to the methane group (2). Similarly peaks at ~37 ppm and ~1.5 ppm
are correlated as methylene (3): the other methylene group (4) is deshielded by
the oxygen atom and so appears at ~63 ppm: ~4.1 ppm. Interestingly, the acetyl
methyl group (6) is upfield of methyl groups (1) in the
13C
NMR spectrum,
whereas the reverse is true for the 1H NMR spectrum. The HETCOR spectrum
clearly shows correlation between peaks at ~20.3 ppm (13C) and ~2.1 ppm (1H)
and, as we know from 1H chemical shift tables, CH3-CO protons resonate around
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~2.1 ppm, the ~20.3 ppm peak can assigned to the acetyl carbon with
confidence.
Hence HETCOR spectra are useful in assigning a nucleus by using
chemical shift data for a (different) nucleus to which it is attached.
HMQC and HMBC Techniques
Like HETCOR, these are 2D NMR spectroscopic techniques that allow
correlations between different nucleus types (e.g. 1H-13C).
HMQC is an inverse H, C correlation method that demonstrates carbon (or other
heteroatom) to hydrogen connectivity by direct C-H coupling. HMBC, on the other
hand, reveals longer range C-H coupling, through 2-4 bonds, including at least
one double bond. The HMBC experiment is designed to suppress 1-bond
correlations (HMQC), but a few are observed in most HMBC spectra; it is a much
more time-consuming technique than HMQC.
The HMQC experiment gives the same information as the older "HETCOR"
experiment, but is more sensitive, so can be done in less time and/or with less
material. This is possible because in the HMQC experiment, the signal is
detected by observing 1H, rather than 13C, which is inherently more sensitive, and
the relaxation time is shorter. For this reason it is known as an inverse
correlation.
The HMQC spectrum of codeine is shown below.
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For example, the contour peak highlighted in red shows that the
13C
NMR peak
indicates that the carbon atom with chemical shift 91.5 ppm is bonded to the
proton with chemical shift 4.8 ppm. The full set of HMQC assignments for
codeine is shown in the table above.
The corresponding HMBC spectrum for codeine is more complex and takes
much longer to acquire. The aromatic part of the spectrum is shown below.
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The red lines in the plot above show correlations from aromatic proton H-8 to
aromatic carbons C-1 and C-6 (both are 3-bond couplings) and a weak
correlation to C-2, which is a 2-bond coupling. The other aromatic proton, H-7,
has correlations to C-2 and C-4, both of which are 3-bond couplings. The green
lines in the plot above show correlations from proton H-9 to carbons C-1, C-3 and
C-4 (all are 3-bound couplings). With the poor digital resolution of the spectrum
in the carbon dimension (512 data points spread over 17 kHz), the peaks for C-3
and C-4 run together because they are barely resolved.
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