2D-NMR spectroscopy

2D-NMR spectroscopy
Part 2
F.D. Sönnichsen
Thursday, Oct 23 2008

The 2D - COSY
Periods in a 2D: Preparation frequency labeling acquisition
Indirect frequency determination =
indirect dimension
mixing
Directly observed =
Direct dimension
Repeat n times,
each time with incrementally increased Δt1
Keep the individual FIDs separate.

Vektor-Diagramm Analyse des 2D-COSYs –die
Frequenzbestimung in der indirekten Dimension
90x
90x
t 1
t 2
n
FID 1:
x
90x
T1=0
T1=τ
90x
Acq
FID 2:
Acq
FID 3:
T1=2τ
Acq
FID 4:
T1=3τ
Acq
Eine Komponente der Magnetisierung wird durch den zweiten Puls in Z-Richtung weitergedreht,
während die zweite Komponente in der transversalen Ebene bleibt. Die Amplitude ist jedoch
verringert, sie ist Sinus-moduliert.
Was passiert wenn wir das Experiment wiederholen, jedesmal mit einer systematisch verlängerten
Delay-Zeit

Sollte wie eine Sinus – Modulation aussehen

Fourier transform
with respect to t 1

Correlating different types of nuclei
The HMQC experiment
Uses the direct coupling of attached nuclei to provide 2D correlation
spectrum. Very sensitive.
The value used for J is typically 145 Hz.
Note: the peak splitting due to the coupling is suppressed via decoupling
during t2, so that we see singuletts in the spectra

Korrelation von unterschiedlichen Kernen
Das HETCOR Experiment
(traditionelle,ursprüngliche
Weg, Protonen mit
HeteroAtomen zu koppeln)
Jetzt selten benutzt
NOTE: Detektion des
Kohlenstoffes
Das HMQC Experiment
(Heteronuclear multiple quantum
coherences)
Benutzt die direkte 1 J-Kopplung
der gebundenen Atome.
Detektiert das Proton. Sehr
empfindlich . Der benutzte Wert
für J ist typischerweise145 Hz.
Note: der Entkoppler –Decoupler
während der Detektionszeit führt
zur Aufnahme von
Singuletts,ungespaltete Signale

Heteronuclear Indirect Detection
Experiments that detect 1 H involved in coupling to an X nuclei ( 13 C or 15 N). Sensitivity
3/2
is enhanced by a factor ⎛ γ H ⎞ where γ is the gyromagnetic ratio.
⎜ γ X
⎟
⎝
⎠
Signal/Noise comparisons
γ H
γ X
⎛ γ H ⎞
⎝
⎜ γ X ⎠
⎟
3/2
Polarization
Transfer for
Direct DET
Nuclei No NOE Full NOE
13 C +1 +3 4 32
15 N +1 Š4 10 306
Indirect
Detection
The much larger sensitivity have lead to the almost exclusive use of indirectly
detected heteronuclear experiments. Note, that these however do not provide
information on quarternary Carbon nuclei!!

The HMBC experiment
MB stands for Multiple bonds
• We have connected neigboring protons (COSY)
• We have connected protons to attached 13 C with the direct 1 J coupling
(HMQC)
• Can we use long range couplings and connect further protons
The HMBC SELECTIVELY observes protons and 13 C that are connected via
small 2,3 J couplings ( of the order of 10 Hz).

Comparison HMQC / HMBC
C-H,
1
J=145,
delay = 3ms
long range C -H,
2
J or 3 J ~ 10Hz,
delay = 50ms

Ipsenol
Note : the arrows indicate
artifacts, i.e. direct couplings
which are not completely
removed. These are 1J
couplings, and can be
identified by their large
splitting. Since we don‘t
decouple during t2, the 1J
couplings constant splits the
signal into a doublett, with a
separation of ~145Hz due to
the 1J-coupling.

More on 2D:
• The principle of 2D NMR can be extended
to 3D, 4D and even higher experiments,
i.e. even more frequencies can be
simultaneously detected. What is the
caveat
• A 1D spectrum takes minutes, a 2D
spectrum hours, a 3D days, a 4D ……

More on 2D:
• Many more 2D experiments can be
created , that correlate the same or further
nuclei with desired specificity
• Most of these experiments correlate nuclei
using coupling constants, i.e. the correlate
through bonds.
• Some also offer the possibility to correlate
nuclei through space

2D -
INADEQUATE
Allows 13 C— 13 C connectivities to be obtained
Incredible Natural Abundance Double
Quantum Transfer Experiment
1. A. Bax, Two-Dimensional Nuclear Magnetic Resonance in Liquids, Delft University Press,
Delft, Holland (1982) pp. 155-174.
2. D. L. Turner, J. Magn. Reson., 53, 259 (1983)
3. D. L. Turner, J. Magn. Reson., 49, 175 (1982)
4. A. Bax and T. H. Mareci, J. Magn. Reson., 53, 360 (1983)
p. 178 - Nakanishi’s text
p. 279 - Silverstein & Bassler & Morril
Sensitivity is extremely low, because
13
C 1.1%
13
C— 13 C 0.01% Correlates double-quantum frequency of 13 C— 13 C
Many variations provide same information, but in different formats
Usually the cross peaks are 2 peaks, representing AX 13 C— 13 C doublets
Pulse sequences are complex and involve long phase cycling to select for desired double
quantum coherences

13C
13C

2D-TOCSY experiment
• Total Correlation Spectroscopy
• Correlates simultaneously all atoms in a
spin system

Same data as COSY (geminal and vicinal couplings), plus RELAY information

2D-TOCSY experiment
Spin system : A, B, D, F
F
Diagonal
TOCSY
COSY
D
B
A
A B D F

TOCSY of a small peptide

Compare the COSY of this peptide

t1
t2
Correlation through space
NOE difference spectroscopy
semi-quantitative measurement of of local proximity
t1
t2

The nuclear Overhauser enhancement
• In contrast to all previous experiments, in which we connected spins through bonds,
the nuclear Overhauser effects enables us to connect nuclei through space.
• This effect is fundamentally different as exchange between the nuclei does not
involve scalar coupling.
• Instead, the direct magnetic coupling (no electrons in between) termed dipolar
coupling is involved, which usually does not have an observable effect in solution (in
contrast to solid state!).
• The nOe can be correlated with internuclear distances and molecular motion.
nOe definition:
η = I - Io
Io
The noe is defined as the normalized intensity change of a resonance line
upon saturating –changing the population of another spin in proximity
NOE = c x r -6
Noes can be measured for protons that are in
close proximity, generally if they are less than
5Å

Noe-background 1
Let’s consider: 2 spins , I and S. They are not coupled ( J=0).
ω 34 , S
βα
4
ω 13 I 1
3
2
ββ
αα
ω 24 I
αβ
ω 12 , S
ω 13 = ω 24 = ω I
ω 12 = ω 34 = ω S
The intensity of the magnetization/ the transition for I and S is given by
the relative populations of these four levels. For I and S, the respective
population differences are:
= 1/2 (P 1
-P 3
+ P 2
-P 4
)
= 1/2 (P 1
-P 2
+ P 3
-P 4
)
and
The irradiation with resonance frequency of S, leads to the equalization of the Populations of the levels for S:P1
= P2, and P3 = P4. At the same time, relaxation will oppose the non-equilibrium situation, leading to
relaxation of the
spins proportional to the following transition probabilities (W I
, W S
, W 2
, W 0
)
W 1 S
βα
W 1 I
ββ
αα
W 1 , I or W I
αβ
W 1 , S or W S
In addition, if the spins are close enough,
the additional two relaxation mechansims (blue arrows)
also exist. These are doublequantum or zero quantum
transistion, involving the spin flip of both spins
simultaneously. Relaxation via these transistion leads
to populationchanges , and thus the sensitivty
enhancement

• It can be easily shown that
NOE=
η =
σ
IS
ϕ
I
=
W2
−W0
2W
+ W + W
I
2
0
For small molecules , W2 is the dominant relaxation pathway leading to positive
NOEs
For larger molecules , W0 is dominant, and one obtains negative NOEs. (intensity
reduction)
Most importantly, the NOE can be quantified and correlated with a distance , since

NOE-experiments
• 1D- steady-state nOe experiment:
– constant irradiation – 1D difference spectrum
• 2D-NOESY experiment:
The 2D-NOESY experiment is the most useful experiments, as all nOe
effect between spins can be measured simultaneously. The nOe is
generated by simultaneous, temporary population changes, which is
called “transient” nOe, not a selective inversion or steady-state
irradiation. The nOe gives rise to magnetization transfer and offdiagonal
peaks, connecting the resonance frequencies of the spins
which couple/ cross relax through space.
90 90 90
d1 t1 tmix t2
Tmix is usually chosen to be between 500ms and 2 sec

z
2D-NOESY: vector model
z
z
90x
t 1
y
y
y
x
x
x
Different y-component
z
z
z
90x
y
y
y
x
Difference in z-component = population change,
causes relaxation, is transfers magnetization,
mixes S with I and vice versa
dIz
dt
= − ϕ 1(
Iz − Io ) − σ IS ( Sz − So
)
x
During tmix transferred to I
During t1: frequency labeled at ωs
IS
During t2 detected with I (ωI) NOE ( ϖ 1,
ϖ 2 ) = ( ϖ S , ϖ I )
I
σ
ϕ

Aphanamol-1
This NOE showed that
the 5-membered ring is
cis to the 7-membered
ring.
15CH 2
5H

Homonuclear
• Through-bond
• COSY (absolute value and
phase sensitive displays, DQF-
COSY)
• TOCSY
• Through-space
• NOESY
• ROESY
Heteronuclear
• Through-bond
• HETCOR
• Long-range HETCOR or
COLOC
• INADEQUATE
• INDIRECT-DETECTION
(HSCQ, HMQC, HMBC)
Pulse Field Gradients (PFG)
NMR- the acronym soup!
(NMR – die Suppenterrine voll Abkürzungen)

Example: Lactose

• 2 sugar units
• 2 separate groups of
coupled spins (termed
spin systems)
• Multiple overlap of the
sugar resonances, and
of the resepective cross
peaks
• Canweusethegood
separation of the
anomeric protons

Lactose - 2D TOCSY

Lactose -ROESY
• Equivalent
Experiment to the
NOESY
• Coupling through
space
• Detects close
proximity of protons
Expansions:

References
J. B. Lambert & E. P. Mazzola, Nuclear
Magnetic Resonance Spectroscopy,
Pearson Prentice Hall, 2004
RM Silverstein, FX Webster & DJ Kiemle,
Spectrometric Identification of Organic
compounds Wiley 2005.