One-Dimensional (1D) NMR Experiments
1D NMR
- General summary
Relaxation – Preparation – Evolution – Mixing – Acquisition
- Relaxation
 signal fully recovers to +z
 should be > 5T1, normally T1 to 2T1 (~1-2 secs.)
- Preparation
select desired information
- Evolution
related to coupling constant (~1/2J)
- Mixing
requires 180 refocusing pulse to phase spectra
usually evolution of through space dipole-dipole relaxation (NOE)
- Acquisition
FID is observed usually with decoupling
One-Dimensional (1D) NMR Experiments
Difference Spectroscopy
-Determine which signals change between different experiments
vary decoupling frequency
 change sample composition (protein-ligand titration)
 change delay times (NOE, coupling)
-Subtract the two spectra
 don’t get perfect cancellation
 Instrument instability
 Bloch-Siegert shift
 Nuclear Overhauser effects

Small change in frequency
Incomplete cancellation
One-Dimensional (1D) NMR Experiments
Decoupling Difference Spectroscopy
-One spectra collected with decoupling off resonance
decoupler set at a frequency far off from any peaks in the spectra
-Second spectra collected with selected decoupling of one peak in the spectra
-Helps deconvolute complex coupling patterns
 repeat for each coupled resonance in the spectra
- coupled spectra give positive signals

- decoupled spectra give negative signals
1H
Difference spectrum (b-a)
1H
spectrum with Decoupler
set on 31P signal of PPh3
1H
Reference spectrum
signals coupled to
31P
One-Dimensional (1D) NMR Experiments
Selective Population Transfer
-Minimize Bloch-Siegert shift
use weak, selective decoupling pulse
 equalizes population of two spin states
 effects population of coupled spin states
-Changes observed from difference spectra

A spins
Normal 1:1 A-X doublet
2dN-dN dN-0
0.5:1.5 A-X doublet after
selective decoupling
1.5dN-dN 1.5dN-0
One-Dimensional (1D) NMR Experiments
Nuclear Overhauser Effect (NOE)
-Dipole-dipole relaxation
-through space correlation (<5Å)

stereochemistry and conformation of molecules
NOE
4.1Å
2.9Å
-Irradiate one nucleus
 intensity of nuclei which are close in space change
 magnitude change depends on nuclei type
 depends on distance between nuclei
Relaxation through
interaction of spin-states
One-Dimensional (1D) NMR Experiments
Nuclear Overhauser Effect (NOE)
Mechanism for Relaxation
• Each nuclei creates a magnetic field that effects other nuclei

Dipole-dipole coupling is described by a unit vector that connects the dipoles
Field at k created by j
• head to tail alignment is lowest energy
 But structures can constrain relative alignment
Magnetic spins are
like bar magnets
Magnitude of dipole-dipole interaction
may come from numerous interactions
One-Dimensional (1D) NMR Experiments
Nuclear Overhauser Effect (NOE)
a)
b)
Important: effect is time-averaged
Gives rise to dipolar relaxation (T1 and T2) and especially to crossrelaxation

Mechanism by which spins return to equilibrium state (aligned
with external magnetic field +z)

Will discuss in detail later in the course
Perturb 1H spin population
affects 13C spin population
NOE effect
One-Dimensional (1D) NMR Experiments
Nuclear Overhauser Effect (NOE, h) – the change in intensity of an NMR
resonance when the transition of another are perturbed, usually by saturation.
hi = (I-Io)/Io
where Io is thermal equilibrium intensity
Saturation – elimination of a population difference between transitions
(irradiating one transition with a weak RF field)
irradiate
bb
ab
N
N-d
X
A
ba
X
aa
N+d
N
A
Populations and energy levels of a homonuclear
AX system (large chemical shift difference)
Observed signals only occur
from single-quantum transitions
One-Dimensional (1D) NMR Experiments
Nuclear Overhauser Effect (NOE)
Saturated
(equal population)
ab
N-½d
saturate
bb N-½d
I
S
ba
I
aa
N+½d
N+½d
S
Saturated
(equal population)
Observed signals only occur
from single-quantum transitions
Populations and energy levels immediately
following saturation of the S transitions
bb
ab
N-½d
W1A
N-½d
W1X
W2
W0
aa
W1X
N+½d
ba
W1A
Relaxation back to equilibrium can occur through:
Zero-quantum transitions (W0)
Single quantum transitions (W1)
Double quantum transitions (W2)
N+½d
The observed NOE will depend on the “rate” of these relaxation pathways
One-Dimensional (1D) NMR Experiments
Nuclear Overhauser Effect (NOE)
bb
ab
N-½d
W1A
N-½d
X
W1
W2
W0
aa
Solomon Equation:
W1X
N+½d
ba
W1A
N+½d
W2  W0
X
hi 
 A 2W1A  W2  W0
Steady-state NOE enhancement at spin A is
a function of all the relaxation pathways
If only W1, no NOE effect at HA
If W0 is dominant, decrease in intensity at HA  negative NOE
If W2 is dominate, increase in intensity at HA  positive NOE
For homonuclear (X=A), maximum enhancement is ~ 50%
For heteronuclear (X=A), maximum enhancement is ~50%(X/A)
One-Dimensional (1D) NMR Experiments
Nuclear Overhauser Effect (NOE)
Intensity of NOE “builds-up” as a
function of time (tm – mixing time)
NOE build-up rate is dependent on correlation time (tc) and frequency
– correlation time: time it takes a molecule to rotate one radian (360o/2p)
– ~10-11 secs. for small molecules
–~10-9 secs. MW:1000 to 3000
–>10-9 secs. MW > 5000
One-Dimensional (1D) NMR Experiments
Nuclear Overhauser Effect (NOE)
Correlation Time
– Debye theory of electric dispersion:
4pa 3 h
tc 
3k T
N – viscosity
T – temperature
a – radius of molecule
k – Boltzman constant
Varying temperature, viscosity or mass of sample will change tc
One-Dimensional (1D) NMR Experiments
Nuclear Overhauser Effect (NOE)
Mechanism for Relaxation
• Dipolar coupling between nuclei and solvent (T1)
interaction between nuclear magnetic dipoles
 depends on correlation time
– oscillating magnetic field due to Brownian motion
– depends on orientation of the whole molecule
 in solution, rapid motion averages the dipolar interaction –Brownian motion
 in crystals, positions are fixed for single molecule, but vary between molecules
leading range of frequencies and broad lines.

Tumbling of Molecule Creates local
Oscillating Magnetic field
One-Dimensional (1D) NMR Experiments
Nuclear Overhauser Effect (NOE)
Mechanism for Relaxation
• Solvent creates an ensemble of fluctuating magnetic fields
causes random precession of nuclei  dephasing of spins
 possibility of energy transfer  matching frequency

2t c
K (v ) 
1  4p 2v 2t c2
Field Intensity at any frequency
tc represents the maximum frequency
– 10-11s = 1011 rad s-1 = 15920 MHz
All lower frequencies are observed
One-Dimensional (1D) NMR Experiments
Nuclear Overhauser Effect (NOE)
Mechanism for Relaxation
Extreme narrowing limit (flat region)
4p 2v 2 
tc ~ 10ns (macromolecule)
tc ~ 10ps (small molecule)
1/tc
Intensity of fluctuations in magnetic field
Proportional to tc (note: different scales)
1
t c2
Relaxation or energy transfers
only occurs if some frequencies
of motion match the frequency
of the energy transition.
The available frequencies for a
molecule undergoing Brownian
tumbling depends on tc.
The total “power” available for
relaxation is the total area under
the spectral density function.
One-Dimensional (1D) NMR Experiments
Nuclear Overhauser Effect (NOE)
Mechanism for Relaxation
• Spectral density is constant for w << 1/tc
tc decreases, wo also decreases and T1 increases
 at 1/tc ≈ wo there is a point of inflection
– W2 falls off first since it is the sum of two transitions
 relaxation rates via dipolar coupling are:

W1A 
3t c
3t c

r 6 (1  w A2t c2 )
r6
3t c
2t c
W0  6
 6
2 2
r (w A  w X ) t c )
r
W2 
12t c
12t c

r 6 (1  (w A  w X ) 2t c2 )
r6
NOE is dependent on the
distance (1/r6) separating the
two dipole coupled nuclei
Important: the effect is time-averaged!
Extreme narrowing limit: 1/tc >>wo then wo2tc2 <<1)
One-Dimensional (1D) NMR Experiments
Nuclear Overhauser Effect (NOE)
Dependence of NOE on tc
• NOE can be positive, zero or negative depending on tc  MW
Zero NOE
positive NOE
negative NOE
Small molecules
Increasing MW
Decreasing tc
Biomolecules, polymers
One-Dimensional (1D) NMR Experiments
Nuclear Overhauser Effect (NOE)
Experimental Aspects of NOE
• 50% NOE is theoretically possible
• In practice, < 5% NOEs are frequently observed
• A number of factors reduces the NOE
 Any relaxation pathway other than dipole-dipole will reduce NOE
– paramagnetic relaxation most common: paramagnetic transition
metal ions or O2  degas sample
 viscous, solvents, MW or presence of solvents lower tc  lower hmax
 NOE builds up by dipole-dipole relaxation
– in small molecules, T1DD > 10 secs.
To differentiate between NOEs
and changes from decoupling, do
not decouple during acquisition
One-Dimensional (1D) NMR Experiments
Nuclear Overhauser Effect (NOE)
NOE Difference Spectroscopy
• selectively irradiate on resonance
intensity will be perturbed for other spatially close nuclei
 subtract spectra with/without irradiation
• Aids in the assignment of the NMR spectra

Strong NOE
must be H3
Irradiate chemically
distinct H7
One-Dimensional (1D) NMR Experiments
Nuclear Overhauser Effect (NOE)
13C Spectroscopy
• nearly always decoupled to enhance signal to noise
lose splitting pattern
 intensities are not reliable parameter to quantify number of carbons
 different values of NOE
 different relaxation times

– Quaternary carbons tend to have very long relaxation times and
are commonly not observed or severely reduced in intensity
• changing when decoupling takes place in pulse sequences can select
between, NOE, 1H coupling and full sensitivity enhancement
Decoupling with NOE
Decoupling with NOE
suppression
No 1H decoupling
One-Dimensional (1D) NMR Experiments
Nuclear Overhauser Effect (NOE)
Decoupling with NOE
suppression
NOE while maintaining
1H coupled spectra
decouple
Decoupling with NOE
One-Dimensional (1D) NMR Experiments
J Modulation (JMOD)
Used to Edit 13C Spectra
• changes the “phase” of C and CH2 signals relative to CH and CH3
C and CH2 point up (positive)
 CH and CH3 point down (negative)
• Maximize sensitivity by complete decoupling and NOE, but maintain spin
system information.

d1 = recycle delay
for relaxation
d2 = 1/J1H-13C
90o
180o
One-Dimensional (1D) NMR Experiments
J Modulation (JMOD)
Aids in NMR Assignments
• Identifies the number of different spin systems
10
presents
• Chemical shifts identifies the types of
functional groups that are present.
4
3
8
1
6
7
9
2
5
6 8
1
2 4
5
3
7
9,10
One-Dimensional (1D) NMR Experiments
J Modulation (JMOD)
Remember Coupling constants are in Hz
(cycles per second)
On resonance
(center of coupling pattern)
13C
• complete cycle is 360o
• each spin moves relative to carrier
(center of spin system) during d2 delay
13CH
•13C singlet:
on resonance doesn’t move during 1/J
•13CH doublet each spin distance from
carrier is J/2  moves 180o in 1/J
•13CH2 triplet:
- center peak on-resonance doesn’t move.
- outer peaks are J from carrier  moves
360o in 1/J
•13CH3 quartet:
- inner doublet are J/2 from carrier 
moves 180o in 1/J. Outer
- doublet are 3J/2 from carrier  moves
540o or an effective 180o in 1/J
13CH
2
13CH
3
180o
decouple
One-Dimensional (1D) NMR Experiments
J Modulation (JMOD)
Phase of the Peaks Differ as a result of
the Different Spin Systems
On resonance
(center of coupling pattern)
13C
• the 180o pulse and the second 1/J delay
allows for refocusing of chemicals shifts that
differ from the carrier position
 rotation is actually dependent on d+J
o
180 reverses direction and refocus
rotation due to d
• 1J13CH ~ 125-170 Hz
 use average J ~ 145 Hz
13CH of alkynes J ~250
 problems with
Hz  behaves like 13CH2
•Decoupler is turned on during second d2 and
acquisition to collapse spins to singlet and
gain NOE sensitivity
• If d2 set to 1/2J, only observe 13C
 difficult  average J  incomplete
cancellation and weak 13C signal
13CH
13CH
2
13CH
3
180o
decouple
One-Dimensional (1D) NMR Experiments
INEPT
Polarization Transfer
• population difference between a and b states is proportional to 
population difference ~ 4x > 13C
1
13C, 13C S/N would
 If this difference could be transferred from H to
increase by a factor of 4.
 Lose of NOE effect
• polarization transfer > NOE effect

1H
One-Dimensional (1D) NMR Experiments
INEPT
Polarization Transfer
• selective 180o on one 1H spin
inverts the 1H a and b spin states
13C population differences are now ±DH instead of +DC

1
 Repeat by inverting other H spin and subtract spectra  in-phase
doublet with 4-fold increase in S/N

Selective 180o
on H1
One-Dimensional (1D) NMR Experiments
INEPT
Polarization Transfer
• Previous described experiment is impractical
need to repeat experiment for each unique carbon present in molecule
• Can achieve the same effect with the INEPT pulse sequence
 simultaneous polarization transfer for all carbons present in molecule
• Common module of multidimensional NMR experiments

90o
180o
90o
180o
90o
d1 = recycle delay
for relaxation
d2 = 1/4J1H-13C
One-Dimensional (1D) NMR Experiments
INEPT
Separation in peaks
indicate triplet (J~145Hz)
J
-1:1 doublet
13CH
INEPT Pascal Triangle
2J
-1:0:1 triplet
13CH
2
-1:-1:1:1 quartet
13CH
3
One-Dimensional (1D) NMR Experiments
INEPT
Decouple INEPT Experiment
• results in selective inversion of one spin in the doublet
• same result as selective polarization transfer
o
 during first d2 = 1/4J each spin moves 45
o 1H refocusing pulse flips spins (would refocus after another 1/4J delay
 180
o
1
 180 X pulse exchanges a and b H spins
– X attached to a are now attached to b and vice-versa
– direction of rotation is reversed
o
o
 During second d2, each spin moves another 45 and are aligned 180 to each other
0
 90 X pulse generates X FID with polarization transfer
 phase cycling of receiver can alternatively add and subtract spectra
Final 1H 90o will place one
spin as +z and the other as –z
Effectively, a selective 180o on
one spin
One-Dimensional (1D) NMR Experiments
INEPT
Effect of INEPT Pulse Sequence on 1H spins
• because spins are 180o to each other, turning on decoupler will cancel spins  no signal
• insert 180o refocusing pulse separated by d3=1/4J delay
180o refocusing pulse
X spin state after
standard INEPT (p6)
Decoupler turned on
X collapse to singlet
One-Dimensional (1D) NMR Experiments
INEPT
Refocused INEPT can Distinguish CH, CH2 and CH3
• selection of d3 as a function of 1/J determines what spins are observed
only 13C attached to 1H are observed
 0.125/J optimal for all positive signal
13CH observed
 0.25/J only
 0.375/J CH2 are anti-phase (negative)
• common component of multidimensional NMR pulse sequences to select desired correlations
• INEPT not commonly used to select spin systems  DEPT
 INEPT is too sensitive to JXH variations

CH
One-Dimensional (1D) NMR Experiments
DEPT
Pulse Sequence of Choice to Edit 13C NMR Spectra
• not possible to use a simple vector model to explain pulse sequence
involves creating multiple-quantum coherence
• variable p3 pulse selects desired spin system and phase
o
 45 pulse: CH, CH2 and CH3 are all positive
o
 90 pulse: only CH signal observed
o
 135 pulse CH and CH3 positive with CH2 being negative
•Addition and subtraction of DEPT-45, DEPT-90 and DEPT-135 can generate spectra that
only contains CH, CH2 or CH3 signals

90o
d1 = recycle delay
for relaxation
d2 = 1/2J1H-13C
180o
ao
One-Dimensional (1D) NMR Experiments
DEPT
(DEPT-45 + DEPT-135) –
DEPT-90
DEPT-45 - DEPT-135
DEPT-90
Normal Spectra
One-Dimensional (1D) NMR Experiments
bb
A
W1
W2
ab
W1X
W0
W1X
DEPT
ba
W1A
aa
Wo,W2: multiquantum,
forbidden transitions
multiple quantum vector
does not change during t
13C
90o creates multiple
quantum coherence
180o pulse refocus
chemical shifts
Anti-phase component
(amplitude function of sin q)
Last 1H pulse
Multiquantum component
(amplitude function of cos q)
One-Dimensional (1D) NMR Experiments
PENDANT
Pulse Sequence of Choice to Edit 13C NMR Spectra
• DEPT does not observe non-protonated 13C atoms
• PENDANT same sensitivity as DEPT
13C, 13CH, 13CH and 13CH
 observes quaternary
2
3
13
 quaternary
C signals are stronger than in JMOD
 C/CH2 are opposite phase of CH/CH3 signals
• PENDANT with chemical shift information generally sufficient to assign 13C spectrum
 ambiguities can be removed with the appropriate DEPT experiment
• Only requires collecting one spectrum
1
13C spectrum
 pointless to acquire simple H decoupled
 replaces JMOD and APT
• Again, simple spin vector diagrams are insufficient to describe pulse sequence
 Creating multiple quantum coherence
90o
d1 = recycle delay
for relaxation
d2 = 1/4J1H-13C
d3 = 5/8J1H-13C
180o
One-Dimensional (1D) NMR Experiments
PENDANT
Signals can be Missing from JMOD, INEPT,DEPT or PENDANT
• relaxation of peaks occur during delays
• worse for broad signals
 due to exchange or quadrupolar nucleus
One-Dimensional (1D) NMR Experiments
INADEQUATE
Detects Carbon-Carbon Coupling
• 13C nuclei only 1.08% abundant
weak satellites on either side of strong center peak
13C is 1.17e-2%
 probability of two bonded atoms both being
• Experiment suppresses strong center peak to detect 13C satellites

Center peak
off-scale
13C
C2
satellites
C1
C4
C3
C5
Identifying 13C-13C connectivity
beneficial for NMR assignment
of complex molecules.
One-Dimensional (1D) NMR Experiments
INADEQUATE
Detects Carbon-Carbon Coupling
• delay (d2) can be set to select 1J13C-13C or longer coupling 13C-13C
•Two-dimensional version (2D) determines 13C-13C connectivity
d1 = recycle delay for relaxation
d2 = 1/4J13C-13C
1H
decoupling on throughout
experiment
One-Dimensional (1D) NMR Experiments
INADEQUATE
d2 = 0.08 sec
J13C-13C = 3 Hz
d2 = 0.0062 sec
J13C-13C = 40 Hz
13C
spectrum
One-Dimensional (1D) NMR Experiments
Summary of Information Present in Some 1D Experiments