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900 MHz NMR
1
Characteristics of Principal Spectrometric Methods
1H-NMR
13C-NMR
0-15 ppm
1-220 ppm
Sample
1-2 mg
10-20 mg
Molecular
formula
Partial
Partial
Functional
group
~ yes
~ yes
Substructure
yes
C-Connection
Scale
MS
IR
50-4000 amu 400-4000 cm-1
< 1 mg
< 1 mg
Yes
No
Limited
Yes
yes
yes
Very limited
yes
yes
No
Very limited
Stereochem. &
yes
regiostereochemistry
yes
No
Very limited
2
ALL SPECTROMETERS HAVE SOME COMMON
ESSENTIAL FEATURES
Electromagnetic
Radiation
Source
SAMPLE
HOLDER
ANALYZER
DETECTOR
RECORDER
(or Computer)
 A source of electromagnetic radiations of the appropriate frequency range.
 A sample holder to permit efficient irradiation of the sample.
 A frequency analyzer which separates out all of the individual frequencies
generated by the source.
 A detector for measuring the intensity of radiations at each frequency,
allowing the measurement of how much energy has been absorbed at each of
these frequencies by the sample; and
 A recorder – either a pen recorder or computerized data station, with a VDU
for initial viewing of the spectrum, with the possibility of manipulation.
3
 NUCLEAR-
study of nuclear spins
 MAGNETIC-
under the influence of
applied magnetic field
 RESONANCE- and to record the resulting
resonance in nuclear spin
through the absorption of
Rf
4
SPECTROSCOPIC TECHNIQUES IN ORGANIC
CHEMISTRY AND THEIR USES
Radiation
Absorbed
Effect on the Molecule
Nuclei placed under the static
NMR
magnetic field change their spins
Radiofrequency
after absorption of radiofrequency
, 25 cm
radiations
Information Deduced
The electronic
environment of nuclei,
their numbers and
number of neighboring
atoms.
5
The first published ‘high-resolution’ proton NMR
spectrum (30 MHz) displaying the proton chemical
shifts in ethanol
6
Fundamental of NMR
Spectroscopy?
7
Angular momentum and magnetic moment
µ = γJ
Where µ is magnetic moment,
J is angular momentum,
and γ is the constant of proportionality called magnetogyric ratio
Now:
Angular momentum is directly proportional to nuclear spin quantum
Number; so,
J = h/2π * I
Therefore,
µ = h/2π * γI
8
Nuclear Magnetic Resonance
Nuclear spin
m = g I * h/2π
m - magnetic moment
g - magnetogyric ratio
I - spin quantum number
h - Planck’s constant
m
I is a property of the nucleus
Mass # Atomic #
I
Odd
Even or odd 1/2, 3/2, 5/2,…
Even
Even
0
Even
Odd
1, 2, 3
I is quantized; it has only certain
values.
9
Spin Quantum Numbers of Some Common Nuclei
The most abundant isotopes of C and O do not have spin.
Element
Nuclear Spin
Quantum No
1H
2H
12C
13C
14N
16O
17O
19F
1/2
1
0
1/2
1
0
5/2
1/2
2
3
0
2
3
0
6
2
(I)
No. of Spin
States
Elements with either odd mass or odd atomic number
have the property of nuclear “spin”.
The number of spin states is 2I + 1,
where I is the spin quantum number.
10
Number of spin states or multiplicity:
If we place an magnetically active nucleus in an external
magnetic field, how many orientations it can adopt. Number of
spin states is given by formula:
m = 2I + 1
For example, for a nucleus with I = ½,
m=2*½+1=2
So it has two spin states (or, orientations, or multiplicities),
They are +1/2 and -1/2.
We can find out individual spin states by using the sequence:
+I, +(I-1), ……-(I-1), -I
11
12
NMR Instrumentation
• An NMR machine is basically a big and expensive FM radio.
Bo
N
S
Magnet
B1
Recorder
Frequency
Generator
Detector
• Magnet - Superconducting. Some electromagnets and permanent magnets still
around.
• Frequency generator - Creates the alternating current (at wo) that induces B1.
Continuous wave or pulsed.
• Detector - Subtracts the base frequency (a constant frequency very close to wo)
to the output frequency. It is lower frequency and much easier to deal with.
• Recorder - XY plotter, oscilloscope, computer, etc., etc.
13
14
A Conventional 60 MHz
NMR Spectrometer
RF (60 MHz)
Oscillator
hn
Transmitter
absorption
signal
RF
Detector
Recorder
Receiver
MAGNET
MAGNET
N
S
~ 1.41 Tesla
(+/-) a few ppm
Probe
15
16
Nuclear Magnets in an External
Magnetic Field (B0)
Bo
Bo > 0
Bo = 0
Oriented in a pattern
Randomly oriented
N
S
Each nucleus behaves like
a bar magnet.
17
18
Orientations of Nuclei with I = 1/2
Nuclei acquire orientation either
Align with the external magnetic field,
Or
Align against the external magnetic field
Bo
Align with
α state
Align against
β state
Which state should be of lower energy and why?
19
NUCLEAR SPIN STATES OF HYDROGEN NUCLEUS
The spin of the positively
charged nucleus generates
m
a magnetic moment vector, m.
+
+
m
+ 1/2
- 1/2
TWO SPIN STATES
The two states
are equivalent
in energy in the
absence of a
magnetic or an
electric field.
20
THE ENERGY SEPARATION DEPENDS ON Bo
- 1/2
DE
= kBo = hn
degenerate
at Bo = 0
+ 1/2
Bo
increasing magnetic field strength
21
EFFECT OF A STRONG MAGNETIC
FIELD
WHAT HAPPEN WHEN A SPIN-ACTIVE
HYDROGEN ATOM IS PLACED IN A STRONG
MAGNETIC FIELD?
….. IT BEGINS TO PRECESS
OPERATION OF AN NMR SPECTROMETER DEPENDS
ON THIS RESULT
22
23
24
Precessional Frequency
When placed in an external magnetic field (B0), a magnetically active
nucleus starts to undergo a particular motion, called precession. The
frequency with which it precesses is called precession frequency. It is
angular frequency (ω0). It can be converted to linear frequency (ν0).
(ω0= 2π ν0)
This is also known as Larmor Frequency
25
Larmor Frequency and B0
What is the relationship between Larmor frequency and applied
magnetic field?
Should Larmor frequency depend on B0?
How?
26
The Larmor Equation!!!
(Angular) frequency of
the incoming
radiation that
will cause a
transition
wo = gBo
gyromagnetic
ratio g
strength of the
magnetic field
g is a constant which is different for
each atomic nucleus (H, C, N, etc)
no = gB0/2π
Here, no is linear frequency (Hz or MHz)
27
Application of an External Magnetic Field
(Putting your sample in the magnet)
z
w0
w0 = g Bo = 2π ν0
m
w0 - resonance frequency
in radians per second,
also called Larmor frequency
n0 - resonance frequency
in cycles per second, Hz
g - gyromagnetic ratio
Bo - external magnetic
field (the magnet)
Bo
m
w
Spin 1/2 nuclei will have two
orientations in a magnetic field
+1/2 and -1/2.
28
E and Bo
If the difference of energy between β and α orientations is ∆E, then
∆E = hgB0/2π
And since,
∆E = hν,
So,
ν = gB0/2π
Using these equations we can calculate the frequency (or, energy) of
the RF radiation which can be absorbed by magnetic nuclei placed in
an applied magnetic field.
29
WHAT IS “RESONANCE” ?
….Absorption of energy by the
spinning nucleus
30
N
w
Nuclei precess at
frequency w when
placed in a strong
magnetic field.
RADIOFREQUENCY
40 - 600 MHz
hn
If n = w then
energy will be
absorbed and
the spin will
invert.
NUCLEAR
MAGNETIC
RESONANCE
NMR
S
31
Nuclear Spin Energy Levels
N
-1/2
unaligned
In a strong magnetic
field (Bo) the two
spin states differ in
energy.
+1/2
Bo
S
aligned
32
Absorption of Energy
quantized
Opposed
-1/2
-1/2
DE
DE = hn
Radiofrequency
+1/2
Applied
Field
Bo
+1/2
Aligned
33
Resonance Frequencies of Selected Nuclei
Isotope Abundance Bo (Tesla)
Frequency (MHz)
g (radians/Tesla)
1H
99.98%
1.00
1.41
2.35
7.05
42.6
60.0
100.0
300.0
2H
0.0156%
1.00
7.05
6.5
45.8
41.1
13C
1.108%
1.00
2.35
7.05
10.7
25.0
75.0
67.28
100.0%
1.00
40.0
19F
267.53
251.7
34
POPULATION AND SIGNAL STRENGTH
The strength of the NMR signal depends on the
Population Difference of the two spin states
Radiation
induces both
upward and
downward
transitions.
resonance
induced
emission
For a net positive signal
there must be an excess
of spins in the lower state.
Saturation = equal populations = no signal
excess
population
35
Boltzmann Excess
Nβ/Nα = e-∆E/kT
Or
Nβ/Nα = 1+ (-∆E/kT)
Or Nα/Nβ = 1 + ∆E/kT)
We know that:
∆E = E – E
Energy of radiation must match with this energy difference if
resonance has to occur. The radiation having energy comparable
to this energy demand falls in the region of radiofrequency(RF).
This is the radiation of very low energy.
Thus:
∆E = hν
So, Nβ/Nα = 1+ (-hν/kT) or Nα/Nβ = 1 + ∆E/kT = 1 + hν/kT
36
Calculation of Nα/Nβ
What would be the value of DE for protons if Bo = 9.4 T.
It is 4 x 10-5 Kcal / mol. What is the frequency, ν, ? (400MHz ?)
Use this equation for energy: ∆E = hgB0/2π
And this for frequency: ν = gB0/2π
Then use this for Boltzmann Excess:
Nα/Nβ = 1 + ∆E/kT
Or
Nα/Nβ = 1 + hν/kT
The Nα/Nβ ratio is only 1.000064.
In one million nuclear spins we have a difference of just 64:
NMR is
very insensitive when compared to UV or IR...
37
MODERN INSTRUMENTATION
PULSED FOURIER TRANSFORM
TECHNOLOGY
FT-NMR
requires powerful computer
38
Energy States for a
Spin 1/2 System
DE = g h Bo = h n
-1/2
Antiparallel
DE
E
+1/2
Bo = 0
Parallel
Bo > 0
Therefore, the nuclei will absorb light with energy DE resulting in
a change of the spin states.
39
Effect of Static Magnetic Bo
Field on Magnetic Moment
40
Net Magnetic Moment
z
w
m
+1/2
Bo
m
-1/2
w
41
The Net Magnetization Vector
w
z
w
one nucleus
x
Mo - net magnetization
many nuclei
z
w
z
y
y
x
vector allows us to
look at system as a whole
x
42
Bloch Vector Model- Boltzmann
Distribution Excess
43
44
Effect of RF Pulse (B1) on Nuclear
Spin and Resonance
45
Relaxation
•Application of RF pulse (lets say 90ox) bring the
bulk magnetization vector to the xy plan.
•The system re-establish the equilibrium by
releasing energy through a process of
relaxations.
>Spin-Lattice (Longitudinal Relaxation) T1
>Spin-Spin (Transverse Relaxation) T2
•This relaxation or decay with time is detected
as FID signals.
46
47
A bit of a tickle and the Protons will Sing
Because the atom is not static the magnets rotate around the external
magnetic field like a gyroscope.
Now that their voices are warmed up and ready to
go, very little is needed to make the protons sing
their song. We don’t use feathers to elicit a
response - we use radio waves !
The spinning nuclei interact with the
radio wave and are knocked out of
their gyroscopic motion. We can
detect the radio wave (energy) lost
as the nuclei return back to their
gyroscopic equilibrium. We do this
by carefully placing a receiver coil
to listen to the song. This is the
NMR signal (in a time format).
PROBLEM 1
Explain behavior of spinning nuclei:
a. In the absence of magnetic field
b. Under the influence of magnetic
field
c. When rf of appropriate energy is
applied to the system
49
Relaxation
50
PROBLEM 2
What will happen if the
radiofrequency pulse is applied for
an unusually long time?
51
PROBLEM 3
From the discussion so far can you
summarize the factors affecting the
population difference between the
lower energy state (Na) and the upper
energy state (Nb) and how is the
population difference related to the
NMR signal strength?
52
PROBLEM 4
There is only a slight excess of nuclei in
the ground state (about 13 in a million
protons at 100 MHz). Would you expect
in the case of a 13C-NMR experiment for
the same population difference to
prevail?
53
PROBLEM 5
Explain what is meant by the Larmor
frequency. What is its importance in an
NMR experiment?
54
PROBLEM 6
What is the magnetogyric ratio, and
how does it affect the energy
difference between the two states and
the nuclear species sensitivity to the
NMR experiment?
55
Chemical Shift
• A naked nucleus and a nucleus surrounded by electrons.
If you place two 1H nuclei (Protons) in a magnetic field (B0), one is
naked ( having no electronic cloud around it, while other having
electronic cloud around it, should both the nuclei receive the same
effect of B0 ?
Why?
• Nuclei differing in electronic cloud around them.
What do you expect for two nuclei having different electronic clound
around them?
56
Shielding and deshielding
• Diamagnetic shielding or diamagnetic anisotropy: what is it?
• Local diamagnetic current: what is it?
The electronic cloud around a nucleus is caused to circulate by the applied
magnetic filed. This generates a counter magnetic field which opposes the
applied field.
57
Beff = B0 – Bi
Here, Beff is B which is actually sensed by the nucleus, and Bi is the
magnetic field showed by the electronic cloud under the induction of
applied magnetic field.
And, Bi is directly proportional to B0
So
Bi = σB0
Here σ (sigma) is the magnetic shielding of the nucleus. Therefor, we
have to modify our equations
∆E = hgB0/2π and ν = gB0/2π
as follows:
∆E = hgBeff/2π and ν = gBeff/2π
Or
ν = g(1-σ)B0/2π
58
Chemical shift scale
Where, ν0 and ν’0 are the operating frequencies in hertz (Hz) and
Megaherts (MHz), and δi is the chemical shift of a nucleus i and its
Units are ppm. Operating frequencies in MHz would be:
If Bo = 7.05 T, = 300 MHz for 1H or 75 MHz for C-13.
If Bo = 11.75 T, = 500 MHz for 1H or 125 MHz for C-13
The chemical shift of a particular proton (1H nucleus):
Reference: TMS or Tetramethylsilane, SiMe4
59
Thus, an NMR signal that absorbs at 300 Hz higher than does TMS at an applied frequency of
300 MHz has a chemical shift of:
Although the frequency depends on the applied field, the chemical shift is independent of it. On
the other hand the resolution of NMR will increase with applied magnetic field resulting in ever
increasing chemical shift changes.
60
Chemical Shift positions
Examples
61
Solution
62
63
Solution2
64
Q1. What would be the chemical shift of a peak that occurs 655.2 Hz
downfield of TMS on a spectrum recorded using a 90 MHz
spectrometer?
Q2. At what frequency would the chemical shift of chloroform (CHCl3,
δ = 7.28 ppm) occur relative to TMS on a spectrum recorded on a 300
MHz spectrometer?
Q3. A 1 GHz (1000 MHz) NMR spectrometer is being developed, at
what frequency and chemical shift would chloroform occur?
----------------------------------------------------------------------------------
65
Answers
• A1.
655.2 Hz / 90 MHz = 7.28 ppm (the chemical shift of chloroform)
• A2.
7.28 ppm x 300 MHz = 2184 Hz.
• A3.
Chemical shift = 7.28 ppm
Frequency = 7280 Hz
66
Chemical Shift Scales for Proton and C-13
• For protons, ~ 15 ppm:
Acids
Aldehydes
Alcohols, protons a
to ketones
Aromatics
Amides
Olefins
Aliphatic
ppm
15
10
7
5
2
0
TMS
• For carbon, ~ 220 ppm:
C=O in
ketones
Aromatics,
conjugated alkenes
Aliphatic CH3,
CH2, CH
Olefins
ppm
210
150
C=O of Acids,
aldehydes, esters
100
80
50
0
TMS
Carbons adjacent to
alcohols, ketones
NMR Spectrum of Phenylacetone
EACH DIFFERENT TYPE OF PROTON COMES
AT A DIFFERENT PLACE
68
Anisotropic effect
69
70
Effects
71
Spin Relaxation
There are two primary causes of spin relaxation:
Spin - lattice relaxation, T1, longitudinal relaxation.
lattice
Spin - spin relaxation, T2, transverse relaxation.
72
Relaxations of Magnetization Vectors
73
Free Induction Decay
The signals decay away due to interactions with the surroundings.
A free induction decay, FID, is the result.
Fourier transformation, FT, of this time domain signal
produces a frequency domain signal.
FT
Frequency
Time
74
Converting FIDs into Frequency
Domain Signals
FIDs are time-domain signals.
Time-domain signals are converted to
conventional frequency-domain signals
by a mathematical process called
Fourier Transformation.
75
FOURIER TRANSFORM
A mathematical technique that resolves a complex
FID signal into the individual frequencies that add
together to make it. ( Details not given here. )
TIME DOMAIN
converted to
FID
COMPLEX
SIGNAL
FREQUENCY DOMAIN
NMR SPECTRUM
FT-NMR
computer
Fourier
Transform
a mixture of frequencies
decaying (with time)
DOMAINS ARE
MATHEMATICAL
TERMS
n1 + n2 + n3 + ......
individual
frequencies
converted to a spectrum 76
Pulse NMR Spectroscopy
 “Pulse”
means
turning
on
the
Radiofrequency for a time period and then
switching it off.
 Pulse of Rf excite all nuclear spins at the
same time.
 Pulse techniques allow the recording
large number of NMR spectra in short
(accumulation of FID transients)
provide a higher signal-to-noise ratio
Ratio).
of a
time
and
(S/N
77
Pulse NMR Spectroscopy
The idea of applying a sequence of
pulses of different phase angles is
of central importance to all NMR
experiments.
78
Pulse Fourier Transform NMR
t
90ºx RF pulse
=
w = g B0
Bo
Bo
A
t
f
w
NMR frequency
Fourier
Transform
Variation of signal
at X axis vs. time
79
PULSED EXCITATION
N
n1
BROADBAND
RF PULSE
contains a range
of frequencies
(n1 ..... nn)
n2
O
CH2 C CH3
n3
S
All types of hydrogen are excited
simultaneously with the single RF pulse.
80
COMPOSITE FID (Free Induction
Decay)- Time Domain Signals
“time domain“ NMR
spectrum
n1 + n2 + n3 + ......
time
81
COMPARISON OF
CW AND FT TECHNIQUES
82
CONTINUOUS WAVE (CW) METHOD
CLASSICAL METHOD
The magnetic field is “scanned” from a low field
strength to a higher field strength while a constant
beam of radiofrequency (continuous wave) is
supplied at a fixed frequency (say 100 MHz).
Using this method, it requires several minutes to plot
an NMR spectrum.
SLOW, HIGH NOISE LEVEL
83
PULSED FOURIER TRANSFORM
(FT) METHOD
FAST
THE NEWER COMPUTER-BASED METHOD
LOW NOISE
Most protons relax (decay) from their excited states
very quickly (within a second).
The excitation pulse, the data collection (FID), and
the computer-driven Fourier Transform (FT) take
only a few seconds.
The pulse and data collection cycles may be repeated
every few seconds.
Many repetitions can be performed in a
very short time, leading to improved signal …..
84
IMPROVED SIGNAL-TO-NOISE RATIO
By adding the signals from many pulses together, the
signal strength may be increased above the noise level.
noise
signal
enhanced
signal
1st pulse
2nd pulse
nth pulse
add many
pulses
etc.
noise is random
and cancels out
85
1D Pulse Sequences
• The simplest one, the sequence to record a normal 1D spectrum, will serve to define
notation:
Vectors:
Mo
y
90y
Short hand:
z
z
x
x
90y
pulse
Mxy
y
acquisition
90y
n
• According to the direction of the pulse, i.e. 90x or 90y (or 90f if we use other
phases) to indicate the relative direction of the B1 field WRT Mo in the rotating frame.
• The acquisition period will always be represented by an FID for the nucleus under
86
observation (the triangle).
Inversion Recovery
• Measurement of T1 is important, as the relaxation rate of different nuclei in a
molecule can tell us about their local mobility.
• Following pulse sequence is used:
180y (or x)
90y
tD
•Analyze after the p pulse:
z
x
y
z
180y (or x)
x
tD
y
•Since we are letting the signal decay by different amounts exclusively under the effect
of longitudinal relaxation (T1), we’ll see how different tD’s affect the intensity of the FID
and the signal after FT.
87
Inversion Recovery (continued)
tD = 0
z
z
x
90y
y
tD > 0
z
x
FT
x
FT
z
90y
y
y
z
z
x
y
FT
y
x
tD >> 0
x
90y
y
• Depending on the tD delay we use we get signals with varying intensity, which
88
depends on the T1 relaxation time of the nucleus (peak) we are looking at.
Spin-Echo Experiment
• Following pulse sequence is used:
180y (or x)
90y
tD
tD
• Analysis after the 90y pulse:
z
y
x
y
tD

x
x
y
y
dephasing
y
tD
x
x
refocusing
180y (or x)
89
Spin-Echoes (continued)
• Back to the <xyz> coordinates:
y
x
z

y
• If we acquire the FID right after the spin-echo sequence, the intensity of the
signal after FT will only be affected by T2 relaxation
• Upon repetition for different tD values, the intensity versus 2 * tD is plotted and
a graph is obtained, but in this case the decay rate will be equal to T2.
90
Chemical Shift in Rotating Frame
91
Coupling Constants
• The energy levels of a nucleus are affected by the spin state of neighboring
nuclei. The two nuclei are thus said to be coupled to each other. This manifests
in particular in cases where we have through bond connectivity:
1
13
H
1
1
H
H
three-bond
C
one-bond
• Energy diagrams. Each spin now has two energy ‘sub-levels’ depending on the
state of the spin it is coupled to:
ab
I
bb
S
J (Hz)
ba
S
I
I
S
aa is called coupling constant (J) and has units
• The magnitude of the separation
of Hz.
• Coupling patterns are crucial to identify spin systems in a molecule and to the
determination of its chemical structure.
92
One-Dimensional Single Pulse
Sequence
93
Spectral Editing by APT (Attached
proton test)
94
95
Polarization Transfer
•
What if we could use the bigger population difference
from sensitive nuclei (1H) and pass it on to the less
sensitive nuclei (13C, 15N), all in a predictable manner?
•
The method is called polarization transfer, and basically
it involves passing the large excess population
(polarization) of the 1H to the insensitive nuclei before we
perturb it.
•
To understand how it works we use the weakly coupled
two spin system energy diagram.
96
Uncoupled 1H / 13C System
b
b
a
B
a
o
1
H
13
C
98
Coupled 1H / 13C System
bb
13
C1
1
H2
ba
1
H1
ab
Bo
13
C1
aa
99
Coupled 1H - 13C System with Gyromagnetic Ratios
-1/2 gH - 1/2 gC
13
C2
1
H2
-1/2 gH +1/2 gC
1
H1
+1/2 gH - 1/2 gC
13
C1
+1/2 gH + 1/2 gC
100
Coupled 1H - 13C System (Add + 1/2 gH + 1/2 gC)
0
13
C
1
+gC
1
H
H
gH
13
C
+ gC + gH
Assume Population in Number:
gH= 4
gC= 1
(Based on Gyromagnetic ratios)
101
Coupled 1H - 13C System with Population
0
Selective
Population
Inversion
(SPI)
13
C2
1
H2
0+1 = 1
1
H1
4 + 0= 4
13
C1
4+1=5
1
H-Signal
Population difference
5-1=4
4-0=4
13
C-Signal
Population difference
5-4=1
1-0=1
102
Coupled 1H - 13C System with Selective
Population Inversion
4
13
C
1
1
1
H
H
0
13
5
1
H - Signal
Population difference
5-1=4
0 - 4 = -4
C
13
C - Signal
Population difference
5 - 0 = +5
1 - 4 = -3
103
PULSE SEQUENCE FOR INEPT
104
Heteronuclear Polarization Transfer
• Also called selective population inversion, or SPI. Again, the intensities of the
lines reflect what we’ve done to the populations of the spin system.
• Lets think of the two experiments in a heteronuclear system (IS, where I is
Carbon and S is Hydrogen):
ab
••
13C
4
2
1,2
bb
3,4
1H
••••
••••
1H
aa
•••••
•••••
1
13C
3
ba
1,3
2,4
I
S
• Here the population differences between the energy levels reflect that we have
a 1 to 4 ratio between 13C and 1H due to the differences in the gyromagnetic
rations. Here is were we start seeing why it may be useful…
105
Heteronuclear Polarization Transfer
• Now we do the same analysis for SPI. If we invert selectively the populations of
1,2, we get the following:
2,4
13C
bb
•••••
3,4
4
•
•
•
•
•
ab
1H
2
••••
••••
1H
aa
••
1
13C
3
ba
I
1,2
S
• we had started with a 13C signal that looked like 1,3
this:
1,3
2,4
I
• By manipulating the polarization of the protons, we obtain an enhancement of
4 in the 13C signal (considering positive and negative signals).
106
O
DEPT
Experiment
HO
O
Me
HO
Me
H
OH
107
One-Dimensional Single Pulse
Sequence
108
One-Dimensional NMR Experiment
109
One-Dimensional NMR Spectrum
of a Protein
110
TWO-DIMENSIONAL NMR
SPECTROSCOPY
111
TIME DOMAINS IN 2D NMR
112
2D NMR Basics
• The first perturbation of the system (pulse) will not be called the
preparation of the spin system.
• The variable tD is renamed the evolution time, t1.
• 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 (t2) as with all 1D experiments.
Preparation
Evolution
Mixing
Acquisition
t1
t2
•t1 is the variable delay time, and t2 is the normal acquisition time. We can envision
having f1 and f2, for both frequencies…
• We’ll see that this format is basically the same for all 2D pulse sequences and
113
experiments.
114
Acronyms For Basic Experiments
Differ Only By The Nature Of Mixing
Scalar Coupling
Dipolar Coupling
Homonuclear
Heteronuclear
COSY
HSQC
TOCSY
Hetero-TOCSY
Multiple
Quantum
HMQC
NOESY
NOESY-HSQC
NOESY-HMQC
116
O
4
N3
6
O
O
O
O
O
3
5
2
1
O
O
O
A
H
N
H
N
OCH(CH 3)2
O
O
O
B
117
Three NMR Spectra of Ethylbenzene
1H-NMR
CH2CH3
{C8H10}
2D NMR
13C-NMR
Source: Professor J. P. Hornak, Rochester Institute of Technology
118
COSY SPECTRA
119
10
Heteronuclear
J- Resolved
Spectrum
5
2
3
OH
7
8
9
120
INADEQUATE
•
13C-13C
Homonuclear shift correlation spectroscopy
(Jcc)
•
Incredible Natural Abundance Double Quantum Transfer
Experiment
•
Chances of finding two 13C nuclei connected to one
another is 0.01X0.01 (one in ten thousand)
•
To detect such weak interactions, it is necessary to
suppress the 100 times stronger signals from molecules
which contain only an isolated 13C nucleus rather than
two adjacent 13C nuclei
121
INADEQUATE (…)
• The 2D plot would look
like this:1
4
2
3
5
6
7
6-7
OH
2
1
3
4
5
6
7
5-6
HO
4-5
2-3
3-4
1-2
• The pseudo-diagonal is calculated from the cross peaks. If we follow all the
correlations, we can basically establish the complete net of 13C
connectivities.
• In other words, we can figure out the carbon skeleton of the complete
molecule. Despite it looks really cool, you need more than 100 mg in 0.5 ml
of solvent...
122
CH3
OH
123
TOCSY
• TOCSY is TOtal Correlation SpectroscopY. Also called
HOHAHA (HOmonuclear HArtmann HAhn).
• During this pulse sequence, after the evolution period t1,
the magnetization is spin-locked (for example by a series of
180` pulses).
• During this mixing time the magnetization exchange
through scalar coupling. During this spin-lock period, the
magnetization behaves as a strongly coupled spin system
and evolve under the influence of a "collective spin-mode".
• In that collective mode, coherence transfer is possible
between all coupled nuclei in a spin system, (even if the are
not directly coupled).
124
TOCSY
• For small mixing period, (e.g. 20-30 msec), COSY type of data can be observed.
• As the mixing period gets longer, correlation with more distant protons can be
observed (e.g. mix=80-100 msec can correlate H1 to H6 in carbohydrate).
• The extent of correlation depends mainly on the length of the mixing period.
125
Pulse Sequence of TOCSY
126
2D-TOCSY SPECTRUM
127
Two-Dimensional TOCSY of Cellobioside
128
2D-Heteronuclear : H-detected
• A large number of pulse sequences have been proposed
for "Reverse-Correlation" experiments which are based
on the detection of proton spectra during t2 - the
detection time - while the heteronuclear chemical shift
is detected during t1 - the evolution time.
• This type of experiments have been proposed due to the
increase of sensitivity that one can expect by detecting
the most sensitive nuclei instead of the low-gamma
nuclei like in HETCOR experiments (as a reminder, the
sensitivity of a nuclei is proportional to the cube of its
frequency!).
130
2D-Heteronuclear : H-detected
• When limited amount of material is available, the direct
detection of Carbon-13 can be almost impossible but the
detection of carbon chemical shift through those 2D "reverse"
techniques is most of the time very easy.
• Good HMQC could be obtained in 2 hr. but direct carbon gave
very noisy spectra after 15 hr.
• There are basically two types of experiments in this category:
• Those that utilize multiple quantum transitions during the
evolution time (like HMQC, HMBC) and those using INEPT
single quantum transitions during the evolution time (like
HSQC)
131
HMQC (Heteronuclear Multiple
Quantum Correlation)
• The HMQC experiment provides correlation between protons and
their attached heteronuclei through the heteronuclear scalar
couplings.
• This sequence is very sensitive (compare to the older HETCOR) as
it is based on proton detection (instead of the detection of the
least sensitive low gamma heteronuclei).
• The basic idea behind this experiment is in fact related to the echo
difference techniques, which is used to eliminate proton signals
not coupled to the heteronuclei. (As a reminder, this cancellation
is made possible by varying the phase of the pulses applied to the
X nuclei (on alternate scans) and by subtracting the two signals as can be seen in the figure below.
132
2D-Heteronuclear : H-detected
133
HMQC Spectrum of Menthol
10
5
4
6
3
1
2
OH
7
9
8
MENTHOL
134
HMQC Spectrum of Neuraminic Acid
135
HMBC (Heteronuclear Multiple Bond
Correlation)
136
HMBC (Heteronuclear Multiple Bond
Correlation)
• The HMBC experiment detects long range coupling between proton and
carbon (two or three bonds away) with great sensitivity.
• The length of the delay can be adjusted to detect relatively large coupling
constants (4-10 Hz) tau = 0.06 s or smaller couplings (2-7 Hz) tau = 0.1 s.
137
HMBC (Heteronuclear Multiple Bond
Correlation)
• The carbon decoupler is never used in this sequence: therefore the
protons displays homonuclear as well as heteronuclear couplings.
• This technique is very valuable to detect indirectly quaternary
carbons coupled to protons
• Detect indirectly quaternary carbons coupled to protons - specially
useful if direct C-13 is impossible to obtain due to low amount of
material available.
• Very useful sequence provide information about the skeleton of a
molecule. It could be an alternative to the 2D-INADEQUATE
experiment (which is so insensitive).
138
HMBC (Heteronuclear Multiple Bond
Correlation)
• Very useful in carbohydrate area as a sequence analysis
tool that provides unique information concerning
connectivities across glycosidic linkages.
• Another area of interest for using HMBC is in the
peptide-protein area - specially when applied to a 15N
labeled protein - It is possible with this technique to get
connectivities between the Nitrogen and the CH proton
of the amino acid of the next residue.
139
HMBC Spectrum
140
Correlation Through Space
Nuclear Overhauser Effect
•
An important tool in structural analysis
•
Based on direct, through space interactions
(dipolar couplings), between nuclear spins
•
Related to nuclear spin relaxation
•
Typically employed during the last stages of
structural investigation
•
Provide information about the
dimensional molecular geometry
three141
Correlation Through Space
Nuclear Overhauser Effect
• Depends upon inter-nuclear separations (spins
close in space)
• The interpretation of NOE measurements
requires care
• Two types of NOE: Steady state (NOE
difference) and Transient (NOESY)
142
Connections Through Space
• Scalar couplings. COSY, HMQC, HOMO2DJ, COSY, and INADEQUATE tell us
about the chemical structure, but nothing about the conformation or
stereochemistry.
• When we saturate a proton in the sample, it will relax by either zero- or
double- quantum processes, giving energy (enhancing) the signals of protons
dipolarly coupled to it (protons close by…). This is the concept of the
nuclear Overhauser effect (NOE):
(***) ab
bb (*)
W1I
W2IS
W0IS
W1S
W1S
ba (*)
W1I
aa (***)
• Relaxation by either W2IS or W0IS will occur depending on the size of the
molecule, actually its rate of tumbling, or correlation time, tc.
143
NOE for Structure Determination
144
Dipolar Coupling and Transition
145
Nuclear Overhauser Effect
146
Nuclear Overhauser Effect
The intensity of the interaction is a function of the distance
between the nuclei according to the following equation.
I = A (1/r6)
r1,2
1H
1H
1
2
r1,3
r2,3
1H
3
I - intensity
A - scaling constant
r - internuclear distance
Arrows denote cross relaxation pathways
r1,2 - distance between protons 1 and 2
r2,3 - distance between protons 2 and 3
The NOE provides a link between an
experimentally measurable quantity, I, and
internuclear distance.
NOE is only observed up to ~5Å.
147
NOE Measurements for E/Z Isomers
148
NOE for Positional Isomers
149
NOE for Endo/Exo Compounds
150
NOE Difference Spectroscopy
•If our molecule has three protons, two of them at a fixed distance (a CH2), we
have:
Hb
C
Ha
Hb
Hc
Ha
Hc
_
=
hab
hac
•This establish that Ha and Hc are closer in space
151
Transient NOE
• One of the problems of steady-state NOE is that we are continuously giving power
to the system (saturation). This works well for small molecules, because W2
processes (double-quantum) are dominant and we have few protons.
• However, as the size and tc increase, other processes become more important
(normal single-quantum spin-spin relaxation and zero-quantum transitions).
• Additionally, there are more protons in the surroundings of a larger molecule, and
we have to start considering a process called spin diffusion:
I
S
Basically, the energy transferred from I to S then diffuses to other nuclei in the
molecule. We can see an enhancement of a certain proton even if it is really far away
from the center we are irradiating, which would give us ambiguous results.
• Therefore, we need to control the amount of time we saturate the system. The
152longer
•we irradiate, the more spin diffusion…
Summary

The NOE enhancement is, among other things,
proportional to r-6, so it allows to determine the
internuclear
distances:
stereochemistry
and
conformation.
 Depending on the size/rigidity (tc) of the molecule,
steady state or transient NOE experiments will give
more accurate results:

Small organic compounds: Steady-state NOE.
 Proteins, nucleic acids,
NOE,
build-up curves.
polysaccharides: Transient
153
 We can do everything at once using NOESY (transient
Magnetic Resonance Imaging
154