Proton NMR

Common types of NMR experiments:
1-H NMR
a. Experiment – High field proton NMR (400MHz).
single-pulse experiment.
Proton NMR
b. Spectral Interpretation
i. Number of multiplets gives the different H-environments
ii. Splitting patterns indicate number of protons on ‘adjacent Cs’
iii. Integration, indicates relative number of types of protons.
iv. Chemical shift (ppm), indicates chemical environment.
One Dimensional H-NMR
1
A H nucleus that is surrounded by higher electron density
will generally come into resonance at lower frequency than a
nucleus surrounded by less electron density.
Thus the number of ‘multiplets’ gives the number of different
H-environments in the molecule.
2
F
Cl
S
Free rotation about sigma bonds
make the nuclei equivalent on the
NMR time scale.
lesser electron density
Low field
High frequency
The ‘mean position’ of resonance of a group of nuclei is termed
the chemical shift, δ, (ppm). δ indicates chemical environment.
higher electron density
High field
Low frequency
A chemical shift a measure of the resonance frequency of a
particular type of nucleus compared to that of a standard
molecule, TMS in 1H-NMR, scaled to the frequency of the
spectrometer and reported as parts per million, ppm.
3
4
Cl
Ratio of the integrated of peaks (peak areas/heights) indicates
relative number of protons in different environs.
F
Cl
S
Peak (multiplet) area ∝ nH generating the signal, however
area peaks are not perfect.
5
6
Hyperfine Splitting; (J-coupling/ scalar coupling)
NMR signals split into multiple peaks when molecules contain
non-equivalent hydrogen atoms that are separated by covalent
(usually no more than three bonds for saturated compounds).
Cl
The multiplets results from the spin-spin coupling
between nuclei, an interaction in which nuclear spins of
non-equivalent adjacent atoms influence each other.
Signal splitting allows the determination of how different Hcarrying atoms are connected in a molecule, because atoms
on adjacent ‘functional’ groups can generally split each other.
The general rule is that a signal will be split into (n+1) peaks
if there are n equivalent (or nearly equivalent) H atoms three
bonds away.
7
8
H H
Cl
CH3
H H
c
a b
Equivalent protons – protons that are having the same
chemical shift and spin coupling.
9
H
H3C
H Cl
CH3
Cl
10
H
H
HH
HH
Splitting (patterns) are indicative of the equivalence (nearly) or
nonequivalence of the adjacent H (environs).
11
12
Cl
HX
I
Cl
I
H
H
I
Cl
I
Cl
I
H
H
A
X
Cl
I
Cl
X
Cl
I
H
X
Cl
H
A
Cl
I
H
H
X
A
stem
JXA
↓
H
A
I
↓HA
Cl
lines
↑ HA
I
↑ H
X
↑
Coupling Constant
I
↓ H
X
I
Cl
HX
A
Cl
↓
HA
H
A
↑
The coupling constant is independent of the applied field.
13
14
HX
HX
HA
↓ ↑
Tree diagram
JXA
Hyperfine splitting arises due to the coupling (transmission
of the spin state information) of the adjacent magnetic
nuclei via bonding electrons. HX feels the spin orientations of HA.
This is a through bond coupling process.
JAX=JXA
The coupling constant is independent of the applied field.15
I
H
H
Cl
↓ ↑
JAX
Coupling Constant
Cl
HA
16
I
In vicinal coupling is the dihedral angle a between the adjacent
C-H bonds and whether or not it is fixed is important. Coupling is
highest when the angle 0° and 180°, and is lowest at 90°.
Bonds that rotate rapidly at room temperature do not have a
fixed angle between adjacent C-H bonds, so an average angle
and an average coupling is observed.
This latter concept is important for the interpretation of 1H-NMR
spectra for alkanes and other flexible molecules.
17
18
Ha
Note the splitting constant J; for ‘groups’ of atoms with spins
coupled, J values are the same!!
#peaks (2)
(3)
(4)
19
The splitting arises because there are n+1 different possible
spin state combinations of n spins, aligned and against the
external magnetic field arising from n equivalent protons
The probability of a molecule having a given set of spins is
proportional to the number of possible spin alignments giving
rise to that spin state.
Peaks in the multiplet pattern conforms to the coefficients
of the Binomial expansion (Pascal triangle - first order
spectra).
21
20
In a first-order spectrum where chemical shift difference is
much larger than their coupling constant (nuclei A and nuclei X),
∆δA-Xνref > 6 JA-X) the interpretation of splitting patterns is
straightforward, multiplets are well separated.
The multiplicity of a multiplet is given by the number of
equivalent neighboring equivalent atoms, n plus 1, (n+1 rule).
Equivalent nuclei do not interact with each other.
The coupling constant is independent on the applied field.
Multiplets can be easily distinguished from closely spaced
chemical shifts.
Peaks in the multiplet pattern conforms to the coefficients
of the Pascal triangle.
22
Splitting by more than one non-equivalent atoms
Splitting by more than one non-equivalent atoms
To predict the splitting pattern use a tree diagram appplying
different couplings applied sequentially.
To predict the splitting pattern use a tree diagram appplying
different couplings applied sequentially.
JAB>JBC
JAB>JBC
General case, a signal will be split into (n + 1)(m + 1) peaks
for an H atom that is coupled to a set of n H atoms with J
value, and to another set of m H atoms with another J.
23
General case, a signal will be split into (n + 1)(m + 1) peaks
for an H atom that is coupled to a set of n H atoms with J
value, and to another set of m H atoms with another J.
24
Complex splitting pattern is determined by the J values
JAB>JBC
(n + 1)(m + 1)
Note the splitting constant J; for ‘groups’ of atoms that are
spin coupled, J values are the same!!
25
(n + 1)(m + 1)
27
Analysis of first order spectral clusters
(n + 1)
26
28
d-q-d
Mark
the peak
andstarting
note the
area
settingthe
thearea
peaks
Examine
thepositions
area ratios
from
anratios,
end. Notice
areas
at to
the
ends
to 1 triangle
patterns.
the
Pascal
values.
Draw ratio
vertical
lines ofCompare
the
same
size
to
indicate
the peak
positions
The ratio values ordain the type of multiplet at this level.
Determine the splitting at this (last) stage. (doublet here)
a-e called levels
29
Reconstruct peak positions and area ratios before
Repeat the procedure until
the process
this last
coupling.end up in a single peak;
i.e. the peak position without hyperfine splitting.
30
d-q-d
A A
H
A
A
H
H
H H
H
A = ?; not a H
31
32
Equivalent protons – chemical shift and magnetic (spin
coupling).
t-t
Chemical Shift Equivalence
Symmetry makes atoms equivalent. Atoms or groups in
identical physical locations relative to a plane of symmetry
or center of symmetry in a molecule are equivalent.
Rapid processes makes atoms equivalent as well.
Area ratio ⇒ Non Binomial
Overlap possible
1:2 ..⇒ 1:2:1?
33
H
H3C
H Cl
CH3
Cl
34
H
H
HH
HH
C2
σ
i
Diastereoscopic – no σ thro’ CH2; nonequivalent
35
36
Rapid Interconversions
Magnetic Equivalence (Spin-Coupling equivalence):
a.
b.
c.
d.
Applicable (to be considered) for chemical shift equivalent
protons only. The chemical shift equivalent protons must have
the same spin couplings to other nuclei to be magnetically
equivalent.
JAX ≠ JA’X
JAX’≠JA’X’
Keto-enol tautomerism
Around partial double bonds
Rings
Rotation around sigma bonds
Non-equivalent protons at o,p positions
Note spectral symmetry
38
Splitting (patterns) are indicative of the equivalence (nearly) or
nonequivalence of the adjacent H (environs).
39
40
Geminal coupling
Non-conformation to the ‘n+1’ rule
Unsymmetrical molecules with restricted bond rotation such as
alkenes or rings, as well as molecules for which stereochemistry
is important (where equivalence do not exist).
Note the splitting pattern for such systems are complex.
41
The dihedral angle between C-H bonds determines the extent of
coupling (J). Therefore the bond rotation is a key parameter.
Where free rotation about the C-C σ bonds exist, H - C bonds
are equivalent (e.g. -CH2- and -CH3) due to the rapid bond
rotation (exception -CH2- is adjacent to a stereo-center).
Where there is restricted bond rotation (alkene or cyclic) H’s
bonded to same C atom may not be equivalent, especially if the
molecule is not symmetrical, the nonequivalent H nuclei on the
same C atom will couple to each other and generating splitting
of the peaks - geminal coupling.
42
AMX, ABX, ABC
a, b, c nonequivalent
43
44
45
46
The tree diagram is useful to identify the spin coupled atoms
Strongly Coupled (Second and Higher order) Spectra
In these spectra the simple rules used to construct multiplets
(area ratios) are no longer valid. This is because the chemical
shift differences between the spins (nuclei A and nuclei B)
are small compared to the couplings ∆δA-Bνref < 8 JA-B.
Peak splitting can range less to more than expected and
unusual relative peak intensities arise; second-order spectra.
The overall effect of strong coupling is the roofing/tilting of
multiplets.
a
Jab
b
Jac
c
Jbc
d
Jde
e
47
48
Exchangeable H atoms
-OH, -SH, -NH
The degree of leaning depends
on proximity of the chemical
shifts of the absorption, and
The strength of the coupling.
Closer ν values and larger
J values leads to greater leaning
of the ‘roof’.
1. OH, NH protons are exchangeable (fast in NMR scale – no
coupling, single peak or merged into background). The
acidic H signal broadens, do not undergo coupling. H-N
would couple to (3J) adjacent protons.
2. OH, SH, NH, H- bonded
a. intermolecular (δ - solvent, concentration, temperature
dependent)
b. intramolecular
3.
14N
(I=1, nuclear quadrupole interacts, lowers lifetime of H
excited states, single broader peak or merged into bkg.)
49
50
d
In solvents where
exchange could
not occur.
51
52
OH
O
Detection of acidic (exchangeable) protons
HOD
O
P
O
O
CH3
In D2O
Acidic protons can be exchanged with deuterium ions.
Mixing a compound with D2O achieves this task. The H-NMR
spectrum of the exchanged product would be absent of the
acidic H peak.
impurity
In CDCl3
The exchanged product is HOD, the H of which appears at
4.7ppm as a single peak.
Solvent Effect: Change of solvent generally changes chemical
shifts slightly due to solvent-solute interactions changing the
electronic environments of atoms.
53
54
Long Range Coupling
nJ; n>3 occur in rigid structures; alkenes, alkynes,
aromatics, stained rings.
W conformation of four sigma bonds.
Appendix F for coupling constants.
55
Areas under multiplets
56
Cl
O
Cl
O
H
O
P O
O
In principle the area under the multiplets are proportional
to the number of protons generating the multiplets.
(2)
(3)
(2)
(1)
(1)
57
58
1-D Experiment
Integration and T1
Usual H-NMR experiment is a single channel experiment
repeated; a single pulse experiment, repeated.
Protons are excited via irradiation (Channel 1) and the same
channel is used to observe the FID of the signal.
FID is then Fourier Transformed to obtain the NMR spectrum.
θ=π/2
Note the FID decay
characterized by a time
constant that is related to
the rate of re- equilibration
p1
59
d1
AQ
d1
60
θ=π/2
θ=π/2
T2 = spin-spin relaxation time
constant (transverse)
T1 = spin-lattice relaxation time
constant (longitudinal)
p1
d1
AQ
d1
d1 > 5T1
AQ
d1
d1 > 5T1
Z
Equilibrium, M0
ω
A collection of spins in a B
attains an equilibrium state
creating a net macroscopic
magnetic moment, M0
p1
d1
Z
T2 = spin-spin relaxation time
constant (transverse)
T1 = spin-lattice relaxation time
constant (longitudinal)
After pulse.
T2
Y
Y
X
X
61
62
θ=π/2
θ=π/2
T2 = spin-spin relaxation time
constant (transverse)
T1 = spin-lattice relaxation time
constant (longitudinal)
p1
d1
AQ
d1
d1 > 5T1
T2 = spin-spin relaxation time
constant (transverse)
T1 = spin-lattice relaxation time
constant (longitudinal)
p1
d1
AQ
Z
T2
d1
d1 > 5T1
Z
ω
Y
T2
T1
X
ω
Y
T1
X
63
64
Signal intensity given by the integration of peaks is proportional
to M0’s the number of a given type of atoms resonating at it’s
frequency.
The ‘decay’ of excited nuclei restores the system to equilibrium.
It takes time to restore, ‘relaxation time’, T1.
However the above fact is dependent on how well the
experiment parameters are set up.
It is imperative that all nuclei relaxes back to equilibrium.
However every type of nuclei do not decay at the same rate.
Pulse experiments (FT-NMR) are repeated, single pulse
experiments, where the nuclei are ‘excited’ and their decay is
followed (during which time an FID is captured) after each pulse.
Therefore, if the ‘next’ pulse in the multi-pulse protocol is applied
before complete relaxation; without giving sufficient delay time
(5T1) between pulses, the signal size in the transformed
spectrum is not proportional to the number of nuclei slow
decaying nuclei (usually less).
All FIDs from the repeated application of a series of pulses
(scans) are summed.
The summed FID is then transformed into the frequency domain
to generate the traditional spectrum.
65
66
Selective Spin Decoupling: Double Resonance
If the spin states of the nucleus/nuclei adjacent to a certain
nucleus is/are flipped between their two spin states very fast,
equalizing the populations then that particular nucleus would
not be able to distinguish (feel) the spin states of the fast spin
flipping neighbors.
Decoupling is done as a two channel experiment.
Channel 1: Excite and observe protons (pulse)
Channel 2: Excite the nucleus to be decoupled (CW)
The resonance peak appears as if the neighbor ‘do not exist’.
Heteronuclear decoupling: different types of atoms involved
(observed atom and decoupled atom type different)
That is, the particular nucleus gets decoupled from the
neighbors.
Homonuclear decoupling: same types of atoms involved
(observed atom and decoupled atom type same)
The result is the elimination of scalar (J) coupling and it results
in the merging of the otherwise spin coupled ‘peaks’.
Further the peaks of the decoupled nuclei vanishes.
67
68
Homonuclear decoupling
π/2
O
P
O
-CH3 protons
irradiated
i.e. methyl Hs decoupled
OH
O
Channel 1
1H observe
O
CH3
p1
d1
AQ
Channel 2
selective
irradiation
-CH269
Heteronuclear decoupling
70
NOE and through space interactions
P
O
31P
irradiated
i.e. P decoupled
OH
O
O
O
Scalar (J) coupling arises due to through bond interactions
of nuclei in close proximity.
CH3
Another type of interaction is dipolar coupling which occurs
through space and is dependent on the direct distance between
nuclei. This interaction manifests in decoupling experiments.
NOE Effect α
-CH-
-CH2-
71
1
r6
The excess energy provided to the molecule via the Channel 2
(much larger than in double resonance experiment, inverts
populations) finds a ‘way’ to dissipate via dipolar coupling to
the ‘nearby’ nuclei. The excess energy shifts population of
spin states from the ‘equilibrium states’. Thus sets up the
need to reestablish the populations.
72
Cl
Methine H
This in turn increases the population of protons low energy
spin states in close proximity, usually ~5 Angstroms from
the decoupled nucleus.
H3C
O
O
O
O
P
O
H3C
Cl
74
Cl
H 3C
O
O
O
O
P
O
H 3C
Cl
methine H
decoupled
Normal spectrum
Selective NOE
A dispersion like output due
to in homogeneity in B
NOE Difference
75
In NOE difference experiments, a 1H is selectively
pre-irradiated until saturation is achieved. During the
pre-irradiation period, NOE buildup occurs at other 1H nuclei
close in space.
Then a 90o pulse is applied, 90o pulse then creates an
observable magnetization, which is detected during the
acquisition period.
π/2
Channel 1
1H observe
CW
d1
p1
AQ
78
The final nOe difference spectra are displayed as the
difference between a spectrum recorded with on-resonance
pre-irradiation and a reference spectrum that has all proton
absorptions at the same conditions but off resonance.
79
80
Nuclear Overhauser Effect:
Consider a two channel experiment where one channel excites
a spin I and the other saturates a spin S.
Assume the spins I and S are close enough to have a
dipole-dipole coupling (the through-space interaction) and there
is no spin-spin coupling (i.e. scalar coupling) among I and S.
This experiment leads to the enhancement of the signal from
spin I. This phenomenon is known as the Nuclear Overhauser
Effect (NOE).
This enhancement is due to the change in relative populations
of the energy levels brought about by the saturation of S spins
and the subsequent relaxation processes.
ββ
W1C
βα
W2
W1H
W1H
W0
αβ
W1C
αα
H 13CO2I S
W2:W1:W0=12:3:2
H 13CO2I S
W2:W1:W0=12:3:2
Note:
The NOE effect occurs through space and is operational within
a ~5A sphere centered around the irradiated nuclei
(“NOE range”).
It does not enhance resonance signals away from “range” from
the irradiated ‘centre’.
NOE allows determination of the proximity of the nuclei to the
irradiated ones, thus is an excellent technique to ascertain
the inter-proton distances/stereochemistry of molecules.
85
Cl
H3C
O
O
H3C
O
P
O
O
Cl
87
NMR signals with high multiplicities (> 5) may have very low
intensities and may be less than the level in the output.
E.g. (CH3)2CHCH2OH, multiplicity = 21.
Multiplet overlapping may give complex peak ‘splitting’.
"Long-range"-couplings (> 3 bond) may be stronger (π bonds).
E.g. CH3COOCH2C=CCH2OH.
Higher order spectra. Relative intensities of peaks of multiplets
changes extensively; usual patterns not followed.
Spin-spin couplings between protons and other magnetically
active nuclei (e.g., 31P, 13C or 19F) leads to additional peak
splitting.
86