1

2D NMR spectroscopy 2D NMR spectroscopy

2D NMR spectroscopy 2D NMR spectroscopy
So far we have been dealing with multiple pulses but a single dimension
- that is, 1D spectra. We have seen, however, that a multiple pulse
sequence can give different spectra which depend on the delay times we
use.
The 'basic' 2D spectrum would involve repeating a multiple pulse 1D
sequence with a systematic variation of the delay time t
D
, and then
plotting everything stacked. A very simple example would be varying the
time before acquisition:
t
D1
t
D2
t
D3
t
Dn
.
.
2D NMR spectroscopy 2D NMR spectroscopy
We now have two time domains, one that appears during the acquisition as
usual, and one that originates from the variable delay.
2D NMR basics 2D NMR basics
The first perturbation of the system (pulse) is called the preparation of the
spin system.
The variable t
D
is named the evolution time, t
1
.
We have a mixing event, in which information from one part of the spin
system is relayed to other parts.
Finally, we have an acquisition period (t
2
) as with all 1D experiments.
Schematically, we can draw it like this:
t
1
is the variable delay time, and t
2
is the normal acquisition time. We can
envision having f
1
and f
2
, for both frequencies.
This format is basically the same for all 2D pulse sequences and experiments.
Preparation Evolution Acquisition
t
1
t
2
Mixing
2
2D NMR spectroscopy 2D NMR spectroscopy
A rudimentary 2D experiment A rudimentary 2D experiment
We'll see how it works with the backbone of what will become the COSY
pulse sequence. Think of this pulses, were t
1
is the preparation time:
We'll analyze it for an off-resonance (
o
) singlet for a bunch of different
t
1
values. Starting after the first / 2 pulse:
90
x
90
y
t
1
t
2
2D NMR spectroscopy 2D NMR spectroscopy
The second / 2 pulse acts only on the y axis component of the
magnetization of the <xy> plane.
The x axis component is not affected, but its amplitude will depend on
the frequency of the line.

90
x
90
x

o

o

90
x
90
x

o

o
A(t
1
) = A
o
* cos(
o
* t
1
)
A(t
1
) = A
o
* cos(
o
* t
1
)
3
2D NMR spectroscopy 2D NMR spectroscopy
f we plot all the spectra in a stacked plot, we get:
Now, we have frequency data in one axis (f
2
, which came from t
2
), and time
domain data in the other (t
1
).
Since the variation of the amplitude in the t
1
domain is also periodic, we can build
a pseudo FD if we look at the points for each of the frequencies or lines in f
2
.

o
t
1
f
2
(t
2
)
t
1
A(t
1
)
2D NMR spectroscopy 2D NMR spectroscopy
One thing that we are overlooking here is that during all the pulsing and waiting
and pulsing, the signal will also be affected by T
1
and T
2
relaxation.
Now we have FDs in t
1
, so we can do a second Fourier transformation in the t
1
domain (the first one was in the t
2
domain), and obtain a two-dimensional
spectrum:
We have a cross-peak
where the two lines
intercept in the 2D map,
in this case on the
diagonal.
f we had a real spectrum with a lot of signals it would be a royal mess. We look it
from above, and draw it as a contour plot - we chop all the peaks with planes at
different heights. Each slice is color-coded depending on the height of the peak.

o

o
f
2
f
1

o

o
f
2
f
1
4
2D NMR spectroscopy 2D NMR spectroscopy
This is data from a COSY of
pulegone...
time time (set of FD's)
time frequency (1
st
FT)
frequency frequency (2
nd
FT)
0 200 400 600 800 1000
t2 pts
500 600 700 800 900
I2 pts
400 500
I2 pts
2D NMR spectroscopy 2D NMR spectroscopy
The same with some real data The same with some real data
Now the contour-plot showing all the cross-peaks:
OK, were the heck did all the
off-diagonal peaks come from,
and what do they mean?
'll do the best can to
explain it, but again, there
will be several black-box events.
We really need a mathematical
description to explain COSY
rigorously.
f
1
5
2D NMR spectroscopy 2D NMR spectroscopy
Homonuclear Homonuclear correlation correlation - - COSY COSY
COSY stands for COrrelation SpectroscopY, and for this particular case in
which we are dealing with homonuclear couplings, homonuclear correlation
spectroscopy.
n our development of the 2D idea we considered an isolated spin not coupled to
any other spin. Obviously, this is not really useful.
What COSY is good for is to tell which spin is connected to which other spin. The
off-diagonal peaks show this, and they indicate that those two peaks in the
diagonal are coupled.
With this basic idea we'll try to see the effect of the COSY 90
y
- t
1
- 90
y
- t
1
pulse
sequence on a pair of coupled spins. f we recall the 2 spin-system energy
diagram:
I S
J (Hz)

S
S
I
I



2D NMR spectroscopy 2D NMR spectroscopy
We see that if we are looking at I and apply both / 2 pulses, (a pseudo
pulse) we will invert some of the popuIation of spin S, and this will
have an effect on I (poIarization transfer).
Since the I to S or S to I polarization transfers are the same, we'll explain
it for I to S and assume we get the same for S to I. We first perturb I and
analyze what happens to S.
After the first / 2, we have the two I vectors in the x axis, one moving at

I
+ J / 2 and the other at
I
- J / 2. The effect of the second pulse is that
it will put the components of the magnetization aligned with y on the -z
axis, which means a partial inversion of the I populations.
For t
1
= 0, we have complete inversion of the I spins (it is a pulse and
the signal intensity of S does not change. For all other times we will have
a change on the S intensity that depends periodically on the resonance
frequency of I.
The variation of the population inversion for I depends on the cosine (or
sine) of its resonance frequency. Considering that we are on-resonance
with one of the lines and if t
1
= 1 / 4 J:
6
2D NMR spectroscopy 2D NMR spectroscopy
f we do it really general (nothing on-resonance),
we would come to this relationship for the change
of the S signal (after the / 2 pulse) as a
function of the I resonance frequency and
J
IS
coupling:
A
S
(t
1
,t
2
) = A
o
* sin(
I
* t
1
) * sin (J
IS
* t
1
)
* sin(
S
* t
2
) * sin (J
IS
* t
2
)
After Fourier transformation on t
1
and t
2
, and
considering also the I spin, we get:
This is the typical pattern for a
doublet in a phase-sensitive COSY.
The sines make the signals dispersive
in f
1
and f
2
.

90
y
J / 2

I

S
f
1

S

I
f
2
2D NMR spectroscopy 2D NMR spectroscopy
Summary of COSY Summary of COSY
The 2D spectrum has cross peaks on the diagonal as well as off the
diagonal.
Everything is doubled, because we have I to S as well as S to I
polarization transfer.
Exactly on the diagonal we see the normal 1D spectrum. Off the diagonal
we see all connected or coupled transitions
7
NMR Assignment NMR Assignment
ExampIe : ExampIe :
IPSENOL IPSENOL
C13 NMR and DEPT C13 NMR and DEPT
CH only CH only
CH and CH CH and CH
3 3
UP UP
CH CH
2 2
Down Down
CC CC
C C- -O O
C C- -C C
8
Unknown: Unknown: psenol psenol
5 aIiphatic carbons: 5 aIiphatic carbons:
2 MethyIs
1 CH
2 CH
2
1 CH 1 CH- -O ( O (deshieIded deshieIded) )
4 4 OIefinic OIefinic carbons: carbons:
2 =CH
2
1 =CH
1 =C
C C
10 10
H H
18 18
O O
Index of H deficiency: Index of H deficiency: C C - -H/2 + 1 H/2 + 1 2 2
In In
13 13
C: All carbons are visible > no symmetry C: All carbons are visible > no symmetry
Counting # protons
attached to Carbon: 17 H 17 H
The one Missing might be
attached to Heteroatom:
OH OH
Proton Spectra: ntegration Proton Spectra: ntegration
1H 1H
4H 4H
Olefinic Olefinic
1 CH 1 CH
2 CH 2 CH
2 2

1H 1H
CH CH- -O O
Aliphatic Aliphatic
6H : 2 Me 6H : 2 Me
Triplet!! Triplet!!
6 H: 6 H:
1 CH, 2 CH 1 CH, 2 CH
2 2
OH OH
9
HETCOR HETCOR
x xo o
o o
CH CH- -O O
Me Me
o o
CH CH
2 2
CH CH
CH CH- -O O
CH CH
Proton Spectra: Expansion Proton Spectra: Expansion
2 x d 2 x d
ddd ddd
ddd ddd
m + m + OH OH dd dd
dd dd Septet? Septet?
dd dd
d d
CH CH
2 2
2 s? 2 s?
d d
10
COSY COSY
o o
CH CH- -O O
Me Me
CH CH
2 2
CH CH- -O O
o o
Me Me
CH CH
2 2
CH CH
CH CH
CH CH
2 2
CH CH- -OH OH
CH CH
2 2
C C
CH CH
2 2
x x
Me Me Me Me
CH o CH o
CH CHCH CH
2 2
o o
x x
o o
Proton Spectra: Analysis Proton Spectra: Analysis
2 2
1 1
3 3
4 4
5 5
6 6
7 7
8 8
6` 6`
1` 1`
1,1` 1,1` 2 2
3a 3a 3b 3b
4 4 5a 5a
5b 5b
7 7 cis cis/trans /trans
8t 8t
8c 8c
6` 6`
H4 : 7 lines, not H4 : 7 lines, not septett septett, , tt tt
11
TripIett of tripIetts/ septett
Septett:
1:6:15:20:15:6:1
Triplett oI tripletts:
1:2:3:4:3:2:1
JJ
1
JJ
1
/2
HMBC HMBC o o
CH CH- -O O
Me Me x x
o o
CH CH
CH CH
2 2
CH CH- -OH OH
CH CH
2 2
C C
CH CH
2 2
x x
Me Me Me Me
CH o CH o
CH CH
CH CH
2 2
o o
o o
CH CH
C C
x x
o o
C C
CH CH
x x
12
NMR Assignment NMR Assignment
ExampIe : ExampIe :
CaryophyIIene CaryophyIIene
oxide oxide
C13 C13- -NMR and DEPT NMR and DEPT
CH only CH only
CH, CH, CH CH
3 3
CH CH
2 2
C CCH CH
2 2
13
C13 C13- -NMR NMR
In oIefinic region: C=CH C=CH
2 2
In aIiphatic region In aIiphatic region
3 3 MethyIs MethyIs
3 CH 3 CH
5 CH 5 CH
2 2
2 quaternary : 60 and 24 2 quaternary : 60 and 24 ppm ppm
1 CH 1 CH- -O (63 O (63 ppm ppm) )
In In
13 13
C: All carbons are visible > no symmetry C: All carbons are visible > no symmetry
C C
15 15
H H
24 24
O O
Index of H deficiency: Index of H deficiency: C C - -H/2 + 1 H/2 + 1 4 4
Counting # protons
attached to Carbon: 24 24
H H
All protons are All protons are
accounted for accounted for
H H- -NMR: NMR: C C
15 15
H H
24 24
O O
3 Methyl 3 Methyl singlets singlets: :
Me Me- -C (quaternary) C (quaternary)
CH2 CH2
No CH No CH- -O O deshielded deshielded
All protons are attached to Carbon. All protons are attached to Carbon.
Oxygen must be ether (no carbonyl Irom
C-13) no alcohol.
In NMR no very deshielded proton or
carbon: ~ epoxyde?
14
HMQC HMQC



CH CH



CH CH
2 2
CH CH
2 2
H H- -NMR: NMR: CH CH
dd dd
q? q?


Methyl Methyl
t t

CH CH
2 2
CH CH
2 2
15
HETCOR: HETCOR: CH CH
2 2

CH CH

H H- -NMR: NMR: assignment assignment

dd dd
q? q?


Methyl Methyl
t t

CH CH
2 2
CH CH
2 2

16
COSY COSY


CH CH
2 2


CH CH
2 2

COSY exp. COSY exp.

CH CH
2 2

17
HMBC HMBC



CH CH
2 2
C C
CH CH
2 2

HMBC exp. HMBC exp.

C C C C
Me Me
Me Me

18

HMBC exp. HMBC exp.

C C

C C
Me Me
Me Me
C C
CH CH
2 2

Structure : Structure : C C
15 15
H H
24 24
O O

C C

All carbons are accounted Ior as well as Hydrogens. Only O O remain

O O
19
NADEQUATE: NADEQUATE:
1 1
J J
C C- -C C

CH CH
2 2

NADEQUATE: NADEQUATE:
1 1
J J
C C- -C C
/ /

C C C C
2 2

C C
2 2

Me Me

C C
Me Me

CH CH
/ /