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The two important parameters obtained from NMR spectra

are;

Two Dimensional (2D) NMR

p
py
Spectroscopy

a. Chemical shift
b. Spin-spin coupling constant
Large molecules with numerous atoms nuclear magnetic
moment does not permit the determination of these
fundamental parameters easily.

Correlation NMR
Some 1D spectra are far too complex for interpretation
because signals overlap heavily e.g. cholesterols, protein
spectra

In
ntensity

1D spectrum of a protein

Chemical shift, ppm

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Nonequivalent proton groups can have nearly the same

chemical shift and/or complex splitting patterns making
1H NMR spectra complicated even for relatively simple
molecules.
The introduction of additional spectral dimensions simplifies the
spectra and provides more information.
Two-dimensional (2D) NMR techniques can be used to solve
such sophisticated structural problems.

Simplification of NMR spectra makes their interpretation

easier and sometimes the only way possible.
The interaction of nuclear spins (1H with 1H, 1H with 13C, etc.)
are plotted in two dimensions
Examples:
COSY information concerning coupled (homonuclear) systems.
HETCOR, HMBC connectivity between protons and carbons.

2-D spectra simplify the complexity arising from overlapping

of peaks.

NOSEY and ROSEY configuration of a molecule.

INADEQUATE constitution of a molecule without 1H-NMR.

David E. Alonso* and Steven E. Warren, NMR Analysis of Unknowns: An Introduction to 2D NMR
Spectroscopy, Journal of Chemical Education 82,1385 (2005)

Common Pulse sequences 1-D: M M 0 sin(2t 2 )e t 2 /T2

1D-HNMR

1-D spectra are plots of intensity vs a frequency

(chemical shift). In 2-D spectra the intensity is plotted as
a function of two frequencies, usually represented as F1
and F2.
There are two ways to present 2D spectra; stack plots
and contour plots.
2-D spectra are presented usually as a contour plot,
where, the intensity of the peaks are represented by
contour lines (recall topographical maps).

1D-13C-NMR
Decoupled

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General Presentation of Correlation Spectra

Two frequency axes. F1 and F2 are Fourier transformed
frequency axis from a time domain signal.
F2
F1

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H-H Correlation Spectroscopy (COSY)

In a COSY experiment, the chemical shift range of the proton
spectrum is plotted on both axes.
The Diagonal of a H-H COSY is its 1-D H-NMR spectrum !

HX

COSY Spectrum
X

F1 co-ordinates of the peaks also correspond to those of the

normal 1-D spectrum (1-D spectrum plotted on the F1 axis)
in H-H COSY.

HA
HX

HX

H Cl

HA

HX

COSY Spectrum

Cl
H

Each peak is specified by the two frequency co-ordinates

(F1, F2). 2-D NMR spectra are always arranged so that the
F2 co-ordinates of the peaks correspond to those found in
the normal 1-D spectrum. (Often 1-D spectrum is presented
on the horizontal F2 axis).

R1 R2 R3

H
X

F1

F2
COSY spectrum of a molecule containing just one type
of protons HX.

F1

F2
COSY spectrum of a hypothetical molecule containing just
two protons, HA and HX, which are not coupled, is shown.

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HA

HX

COSY Spectrum

In 2-D spectra the idea of a multiplet consists of an array of

individual peaks often forming of a square or rectangular outline.
Multiplets form a square or rectangle with two vertices on the
diagonal.

JAX
A

R3

HA

H H

HX

F1

HA

R1 R2

COSY spectrum of a hypothetical molecule containing just

two types of protons, HA and HX, which are coupled is shown.
COSY spectrum has some symmetry about the diagonal, F1=F2,
which shown above.

In a homonuclear COSY spectrum, the presence of a cross-peak

multiplet F1 = A, F2 = X indicates that the two protons A and X
at chemical shifts A and X are scalar coupled.

HX

F1
F2
F2
Diagonal multiplets
centered around same
F1 and F2.

Cross-peak multiplets
centers around different
F1 and F2 co-ordinates.

H-H COSY

If there had been no coupling, their magnetizations would not

have given rise to off-diagonal peaks.
COSY spectrum shows which pairs in a molecule are
coupled (thro bond coupling, hence connectivity).
Recognition of the preceding fact is the essence for the
analysis COSY spectra.
From a single COSY spectrum it is possible to trace out the
whole coupling network in the molecule.

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Prototype pulse sequence 2D NMR

(/2)x

Preparation

M [M 0 sin(2t1 )e t1 /T2 ] sin(2t 2 )e t 2 /T2

(/2)x

Evolution, t1

Prototype pulse sequence 2D NMR

Detection, t2

Mixing time

M M 0 sin(2
( t1 ))e t1 /T2

Data acquired at the

end of the an acquisition
(after acquisition pulse)
is labeled with the time
variable labeled t2.

The generation of a 2D experiment:

In addition to preparation and detection (done in the 1D
experiment) the 2D experiment has an indirect evolution
and a mixing sequence, time t1.
a. Do something with the nuclei (preparation)
b. let them precess freely (evolution) t1
c do something else (mixing) t1
c.
e. and detect the result (detection, of course).

The basic 2D spectrum would involve repeating a multiple

pulse 1D sequence with a systematic variation of the evolution
and mixing times, t1, and then plotting Fourier transformed FID.

This generates two time domains, one of which, t2, is the

acquisition time that appears during the acquisition as usual,
and the other time domain originates from the variable delay
part, t1.

After preparation the spins can precess freely for a given time t1.
During this time the magnetization is labeled with the chemical
shift of the first nucleus.

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frequency data (FID) in one axis (f2, from t2),

t1
A(t1)

t1=2t

Time domain data in t1. It is

periodic; a pseudo FID created for
each of the frequencies in f2.

t1=t
t1

Decay not shown.

Note all tops form one FID, ..

t1=0

f2 (t2)

t2

t1

Appearance:
O

On the 2D-NMR spectra an additional chemical shift

(homonuclear or heteronuclear) is recorded on the third axis.
t1

t1
stacked plot
http://www-keeler.ch.cam.ac.uk/lectures/understanding/chapter_7.pdf

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f1

f2

COSY:
In a real molecule where J coupling exist, during the mixing
magnetization can be transferred from one nucleus to a
second one. Mixing sequences utilize two mechanisms for
magnetization transfer, namely scalar coupling or dipolar
interaction (NOE).

Two dimensional FT yields the 2D spectrum with two

f
frequency
axes. If the
th spectrum
t
is
i homonuclear
h
l
(signals
( i
l off
the same isotope - say 1H, are detected) the spectrum
would have a characteristic symmetric topology.

1D- double resonance experiment that is often used to find

relationships between protons, the protons are irradiated one
by one.
COSY generates all information from a series of double
resonance experiments in one output (2D spectrum).
The pulse sequence for a COSY experiment contains a
variable delay time as well as an acquisition time. The
experiment is repeated with different and incremented delay
times, and the data collected during the acquisition are stored
in the computer. The value of the delay time is increased by
regular, small intervals for each experiment, so that the data
that collected consist of a series of FIDs collected during the
acquisition, each with a different value of delay time.

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In the COSY experiment, the magnetization is transferred by

scalar coupling.

Signals on the diagonal divides the spectrum in two equal

halves. Signals symmetrical to the diagonal called cross
signals (peaks).

In the COSY spectrum of a molecule where all possible

off-diagonal peaks are generated; the result is a complete
description of the coupling partners in a molecule.

The diagonal results from contributions of the magnetization

that has not been changed by the mixing sequence.

S
Sometimes
ti
the
th coupling
li b
between
t
protons
t
th
thatt are more than
th
Three chemical bonds apart can be seen.

Pulegone

The cross signals originate from nuclei that exchanged

magnetization during the mixing time. They indicate an
interaction of these two nuclei. The cross signals contain
the information of 2D NMR spectra.

Contour plot

time - time

200

400

600

800

t1

1000
pts

t2

t2

time - frequency
500

600

700

800

t1

900
pts

f2

f2
f1
f2
frequency - frequency
f2
f2

400

500
pts

http://tonga.usp.edu/gmoyna/NMR_EN/NMR_lectures.html

f1

http://tonga.usp.edu/gmoyna/NMR_EN/NMR_lectures.html

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1
Stick diagram;

d-d

Each circle represents the

center of the multiplet.

3
1 axis

4 types of H

1-H NMR
Spectrum

H4 H3H1

H2 no coupling
2 axis

C3H8O; U = 0

C3H8O: COSY
4

CH3 CH2 CH2 OH

CH3 CH2 CH2 OH

CH2 OH

1
CH3

CH2

Pick a good starting point

peak label
4
3

2H

2H

3H

1H

H1 H2 H4
Pick multiplet(s) that can be assigned to a group atoms.

Science Tools

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CH2 - O

C5H8O2
CH2

U = 5 8/2 +1 = 2

C5H8O2

b
a

-O
d

a
b

a
2

c
Expansion show
ba coupling
|
-O-C=O; ester

CH2 - O

COSY spectrum
ba

C8H16O : U = 8 16/2 + 1 = 1
U = 2, -CO2 group accounts for 1,
Therefore other is a ring.

3H
2H

cba

CH2 CO CH2

cbad
1D HNMR; four CH2
CH2 - O
No double/triple bonds
Cyclic structure of 4Cs

13C NMR
No equiv. C
CH2O
O-C=O

4H

3H
2H

2H

b CH2
c CH2

a CH2

C=O

d CH2
O

Ketone
C=O

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Me-1

C8H16O

2 3

COSY spectrum
Me1 2 - 3 4 - 5
7 Me8

U = 1; 2 Me groups, 3 CH2 groups

and 4 aliphatic Hs.
CO group accounts for U=1.

Two spin systems

1D HNMR;; five CH2

2 off-peaks
on same line overlap

CH2 CO CH2

Me1 CH22 CH23 CH24

13C NMR
No equiv. C
C=O, ketone; accounts for U=1
CH27/5

CH25
C=O
CH27

Me-8

Me8

C11H20O4 U=2

H-NMR
X2

2H

CH2 CH3
2H

3H

3H

4 multiplets; area 3:3:2:2

Total H atoms = 20
Symmetrical structure

O CH2 CH3

Chemical shifts: two methylene groups OCH2CH3

and
d CH2CH3.
CH2CH3 Th
Thatt ttakes
k 10 H atoms.
t

O-C

13C NMR
6 types of C; also OC=O and OC

O-C=O

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C11H20O4

methyl
CH2 CH3

O CH2 CH3

Ipsenol spectra explanation.

O = C O CH2 CH3
CH3 CH2

CH2 CH3

O = C O CH2 CH3

HO

4 CH2
2 aliph., 2 olef.
Two spin systems

2 CH
2 spin systems

Ipsenol
C10H18O

Ipsenol
C10H18O

HO
1

1 1

2 11

2 1 1

DEPT90 CH
DEPT135 CH, CH3
CH2

13C NMR

13

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Point of entry
distinctive peak

DQFCOSY

DQFCOSY cleans some clutter on COSY by removing

some high intensity (methyl) peaks.
HO

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Point of entry
distinctive peak

Point of entry
distinctive peak

HO

HO

Point of entry
distinctive peak

Ipsenol spectra explanation.

= deshielded
OH deshielded

Lowest,

Diastereoscopic
Geminal, 1 vicinal

HO

Diastereoscopic
Geminal, 2 vicinal

HO

Diastereoscopic
methyls, 1 vicinal
Highly coupled, overlapped with OH.

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O
O

singlet

http://www.chem.ucalgary.ca/courses/351/Carey5th/Ch13/ch13-2dnmr-1.html

http://www.chemistry.ccsu.edu/glagovich/teaching/316/nmr/cosy.html

HETCOR- Heteronuclear Chemical Shift Correlation

COSY

1H-1H

COSY (COSY)
The information on how the H and H are
coupled is gleaned from the contour peaks.

13C-1H

COSY

C Detected (HETCOR)
H Detected COSY (HMQC)
H- Detected Long Range
(HMBC)

The information on how the H are C are

correlated is gleaned from the contour peaks.

HETCOR gives the correlations between protons and other

nuclei such as 13-C or 15N. Two versions exist absolute value
HETCOR and phase sensitive HETCOR. A related experiment
is the HMQC experiment
H-H
H
H couplings are removed here
here. Variations of the HETCOR
can show only CH, or CH and CH3 positive and CH2 negative.
The experiment encodes the proton chemical shift information
into 13-C signals that are observed. It generates cross peaks
for all protons and 13C nuclei that are connected by a 13C-1H
coupling over one bond.

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HETCOR

HMQC

1J

HC

= 145 Hz.

Correlates 13C directly attached to H , large 1JCH couplings

(polarization transfer) and the frequency domains are from
different nuclei . F2 is 13C and F1 is 1H. Therefore no
diagonal symmetry.

-OCH2-

1J

HC

= 145 Hz.

Correlates 13C directly attached to H, and the experiment

is H detected. Long range couplings eliminated.

-CH3

ethyl 2-butenoate

O
O

(HETCOR spectra recorded by D. Fox, Dept of Chemistry, University of Calgary on a Bruker Advance DRX-400 spectrometer

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O
O

CH3
H

H3C

Ethyl Crotonate

O
O

CH3
H3C

Ethyl Crotonate
O
H

O
CH3

H3C

Ethyl Crotonate

Diagonal leads to no
information.

http://www.tecmag.com/pdf/HETCOR.pdf

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HMBC

1/2J can be optimized

f different
for
diff
t
coupling constants
Correlates 13C with 2-bond and 3-bond couplings to H, and
the experiment is H detected. Interpretation more difficult
Because of both 2,3-bond (sometimes 4-) correlations are
present.

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NOESY
1H-1H NOESY (Nuclear Overhauser Effect SpectroscopY)
signals the signals arising from protons that are close to each
other in space regardless of bonding. A NOESY spectrum
arises from through space correlations via spin-lattice relaxation.
Provides a means to establish 3-D structural relationships of a
molecule.

The COSY cross peaks that would arise from the experiment
are also present in the NOESY spectrum (effectiveness; r-6)
The peaks additional to COSY peaks are the NOE enhanced
peaks.

NOESY also detects chemical and conformational exchange

(EXSY). It is a homo-nuclear 2D plot, with diagonal as the
normal 1-D spectrum and projections on each axis. Information
gleaned from the "cross-peaks", which appear at the
coordinates of 2 protons which have an NOE correlation.

NOESY spectrum of codeine

Expansion of the up-field region;

http://www.acornnmr.com/codeine/noesy.htm

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N-Phenylacetamide

8 - 7, 12
7 - 18, 18'
3 - 5, 10
5 - 11, 16,
18'
9 - 10, 17,
17'
10 - 16
11 - 18, 16,
14, 18'
18 - 13, 18'
16 - 14, 17
13 - 14, 17,
17'
13' - 17, 17'
17 - 17'

~2

~2

' indicates the more up-field of geminal CH2 protons

NOSEY: The NOE enhanced peaks (only) for A and B

HNMR: Simulated Spectrum N-Phenylacetamide

H
N
O

A
B

N-phenylacetamide

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