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Report
Chemistry 125: Lecture 61
March 26, 2010
NMR Spectroscopy
Through-Space Coupling,
Decoupling & Correlation
This
For copyright
notice see final
page of this file
A spin = 1/2 nucleus is on perfectly
frictionless bearings in a molecule,
oriented by the magnetic field.
Proton Decoupling
25 MHz
100 MHz
(in frame rotating
at 100 MHz)
13C
C13 NMR
spectrum
irradiate H (100 MHz)
and pulse (25MHz)
to observe C13
H
H NMR
spectrum
or
or
J ~ 125 Hz
H up
H average
H down
C13 up
C12
C13 down
Observe 13C while decoupling 1H at various powers.
40 db
CDCl3
CH2
CH
CH
C
(inverse log measure of rf power)
CH2
CH3
Observe 13C while decoupling 1H at various powers.
40 db
(inverse log measure of rf power)
Observe 13C while decoupling 1H at various powers.
20 db (inverse log measure of rf power)
Observe 13C while decoupling 1H at various powers.
15 db
(inverse log measure of rf power)
Observe 13C while decoupling 1H at various powers.
10 db
(inverse log measure of rf power)
Observe 13C while decoupling 1H at various powers.
5 db
(inverse log measure of rf power)
Observe 13C while decoupling 1H at various powers.
2 db
(inverse log measure of rf power)
Observe 13C while decoupling 1H at various powers.
1 db
(inverse log measure of rf power)
NOE
(Nuclear Overhauser
Enhancement)
CH
C
RF excitation of a
CH2
nucleus strengthens
the signal from
CH nearby nuclei.
Bad for
integration
Good for
determining
structure
(see below) CDCl
3
CH2
CH3
H1
F19
99.98%
MHz
P31
C13
1%
O17
6%
Precession
Frequencies in
Magnetic Field
of ~23.5 kGauss
Proton-Decoupled 13C NMR Assignments for
the Artificial Sweetner Neotame Monohydrate
18-20
O
O
1
H +
16
15
H N
H
2 3 4 N
17 O
5
One peak per
carbon, pretty
14 well spread out
13 O
O
Only 1% of 13Cs
6
have a 13C neighbor
in the same molecule.
C=Ono 13C-C13arom
Why
C splitting?
7-12
C-X
e-neg
Calkane
Prof. Eric Munson, Kansas Univ.
Power of Correlation:
Dilute
13C
Double Labeling
2-D NMR
2-D Chromatography
Double Labeling
Introduction:
Lanosterol Biogenesis
Cf. Frames 6-13 of Lecture 52
and Sec. 12.13 pp. 554-562
Squalene
+
+
+
HO
+ O+
H
+
+
Squalene
H 3C
+
H
H H
+
CH3
H 3C
CH
3
H+
H
+
+
CH3
+H
CH3
CH3
CH3
Lanosterol
HO
H 3C
CH3
(source of cholesterol
& steroid hormones)
Squalene
H 3C
Cute Story
H H
+
CH3
+
3° H
3° H
+
CH3
3° CH
3
H 3C
CH
3
CH3 +
Is it
True?
+
CH3
H+
H
3°
3°
Lanosterol
(Wait for NMR)
HO
H 3C
CH3
(source of cholesterol
& steroid hormones)
Squalene
Single Label Enrichment
H 3C
OPP
13C
H
CH3
H
Label
CH3
CH3
H 3C
CH3
Enriched Peaks
(100x stronger than
natural-abundance
peaks)
Lanosterol
HO
H 3C
CH3
Squalene
Single Label Enrichment
H 3C
OPP
13C
H
CH3
H
Label
CH3
CH3
H 3C
CH3
Enriched Peaks
Lanosterol
HO
H 3C
CH3
Squalene
H 3C
OPP
13C
H
CH3
H
Double Label
CH3
CH3
H 3C
CH3
Lanosterol
HO
H 3C
CH3
Squalene
Few single presursor molecules
have any C13 label, but those
HC
that are labeled have two C13s. 3
OPP
Double Label
DILUTE !
13C
CH3
HO
H 3C
CH3
H
CH3
H
CH3
The dilute double label
CH3
experiment enhances the
H 3C
These13 are both labeled,
same
12 labeled,
C peaks but
as the
both
not
in
the
same
molecule
single
label
experiments,
in the
same
molecule
but only 8 of them show
spin-spin splitting (because
their C-C bond stays intact).
Strongly confirms the rearrangement scheme.
Double-Doublet (proton decoupled)
13C-13C splitting (neighboring 13Cs)
Proves
that they
as a unit.
Power
ofentered
Correlation
Power of Correlation:
Dilute
13C
Double Labeling
2-D NMR
2-D Chromatography
Less-congested, off-diagonal peaks appear when “tickling”
one signal on the diagonal enhances another.
is within 6Å of NHs
at d 8.9, 8.3, 8.25, 7.7
NH at d7.25
1H
to 1H
Correlation by NOE
(through-space
magnetic interaction)
proximity
(< 6Å) of
R H
O
H
N
Identify NH with
N
amino acid by
H
O
H
coupling through R
protons
CH to R
in protein
polymer
Narrow range; mostly HN-C=O protons
With Molecular Mechanics Constraints
gives 3-D Structure (without crystal!)
H3C CH3CH3
H3C
CH3
+
CH3
A HBC
A
A D
3
H 3C
CH3
H3C
Methide Shift:
1-2 (as shown)
or 1-Anywhere?
A
CH3
C
H 3C
CH3
+d+
CH3
1H
vs. 1H
Correlation
in TIME
C A
+
+
+
CH3
C
D
(Range of peaks is 150 Hz in 60 MHz spectrometer = 2.5 ppm.)
d+
+
H3 C
CH 3
B
B
CH3 D
0.3 sec 40°C
B
B C
Note: ppm scale is slanted and
"wackbards". The protons in methyls
C and “2-Dimensional”
D are near + charge (see resonance
C D
structures), thus deshielded
NMR from lack of electron
density, and appear furthest to right - at highest d.
d++
C CH3
CH3
H3 C
A
+
d+
B
d+
CH3
d+
CH3
H3 C
C
D
CH3 B
"3-D" Version of contour plot on previous slide
Power of Correlation:
Dilute
13C
Double Labeling
2-D NMR
2-D Chromatography
Developed with
CHCl3 : MeOH : C6H6 (25 : 5 : 3)
Developed with
EtOAc : 96%EtOH : H2O
(80 : 15 : 5)
Thin Layer
Chromatography
of partially
purified extract
of brown algae
looking for
ecdysteroids
http://www.chromsource.com/
books/Milestones-TLC.pdf
better resolution
in
2-D
Thin Layer
Chromatography
of partially
purified extract
of brown algae
looking for
ecdysteroids
("3-D" because
ecdysone
spots turn
turquoise
after vanillin/
H2SO4 spray)
http://www.chromsource.com/
books/Milestones-TLC.pdf
End of Lecture 61
March 26, 2010
Copyright © J. M. McBride 2010. Some rights reserved. Except for cited third-party materials, and those used by visiting
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J. M. McBride, Chem 125. License: Creative Commons BY-NC-SA 3.0

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