CH437 CLASS 17
NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY 8
Synopsis. Two-dimension NMR spectroscopy: COSY and HETCOR.
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 COSY and H-non-H correlation spectroscopy,
known as 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. 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.
The Basic COSY 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
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  = 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
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 ketone 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 H b.
The same thing can be done for cross peaks B and C: this will show H c, 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).
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
the methine proton H3 can also be seen, even though the H3 contour on the
diagonal line is obscured by overlaps.
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
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 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
cross peak B (1H: ~1.0 ppm,
13C:
13C
spectrum (y axis). Similarly,
~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
~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.
The final example HETCOR spectrum is of 4-methyl-2-pentanol (below)
Here we see two spots that correspond to the two nonequivalent methylene
protons at carbon (3) (~1.2 and ~1.4 ppm in the 1H spectrum both correlate with
the ~48 ppm peak in the
13C
spectrum. In the proton spectrum, a pair of doublets
centered at ~0.8 ppm correlate with two peaks in the carbon spectrum (~22 and
~ 23 ppm), indicating that the methyl groups (5) and (6) are nonequivalent. This
is all in accord with the fact that carbon atom (2) is stereogenic, making both
carbon (3) protons and the pair of methyl groups (5) and (6) diastereotopic.
Newman
projection formulas
relationship.
of
4-methyl-2-pentanol (below) show this