One-Dimensional (1D) NMR Experiments

1D NMR
- General summary
Relaxation – Preparation – Evolution – Mixing – Acquisition

- Relaxation
 signal fully recovers to +z

 should be > 5T , normally T to 2T (~1-2 secs.)

1 1 1
- 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
1
H signals coupled to 31P

Difference spectrum (b-a)

1H spectrum with Decoupler

set on 31P signal of PPh3

1
H Reference spectrum
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

N-N N-0

0.5:1.5 A-X doublet after

selective decoupling

N-N N-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
Relaxation through
-Irradiate one nucleus interaction of spin-states
 intensity of nuclei which are close in space change

 magnitude change depends on nuclei type

 depends on distance between nuclei

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 Magnitude of dipole-dipole interaction

like bar magnets may come from numerous interactions
One-Dimensional (1D) NMR Experiments

Nuclear Overhauser Effect (NOE)

a) Important: effect is time-averaged
b) Gives rise to dipolar relaxation (T1 and T2) and especially to cross-
relaxation
 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, ) – the change in intensity of an NMR

resonance when the transition of another are perturbed, usually by saturation.

i = (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
 N-
A X
N
  N
X N+
 A
Observed signals only occur
Populations and energy levels of a homonuclear from single-quantum transitions
AX system (large chemical shift difference)
One-Dimensional (1D) NMR Experiments

Nuclear Overhauser Effect (NOE)

Saturated
(equal population)
saturate
 N-½
S I
N-½ N+½
 
I N+½
 S Saturated Observed signals only occur
(equal population) from single-quantum transitions
Populations and energy levels immediately
following saturation of the S transitions

N-½

W1A W1 X Relaxation back to equilibrium can occur through:
N-½ W2 N+½ Zero-quantum transitions (W0)
 
W0 Single quantum transitions (W1)
W1X W1A Double quantum transitions (W2)

N+½

The observed NOE will depend on the “rate” of these relaxation pathways
One-Dimensional (1D) NMR Experiments

Nuclear Overhauser Effect (NOE)

N-½ Solomon Equation:


W1A W2 W1X X W2  W0

N-½

N+½ i 
W1 X
W0
W1A
 A 2W1A  W2  W0

N+½ 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 (m – mixing time)

NOE build-up rate is dependent on correlation time ( c) and frequency

– correlation time: time it takes a molecule to rotate one radian (360o/2)
– ~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:

4a 3 
c 
3k T
N – viscosity
T – temperature
a – radius of molecule
k – Boltzman constant

Varying temperature, viscosity or mass of sample will change c

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

2 c
K (v ) 
1  4 2 v 2 c2
Field Intensity at any frequency

c 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)
c ~ 10ns (macromolecule)
c ~ 10ps (small molecule)
1/c
Intensity of fluctuations in magnetic field
Proportional to c (note: different scales)
1
4 2 v 2 
 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 c.
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/c
 c decreases, o also decreases and T1 increases
 at 1/ ≈  there is a point of inflection
c o
– W falls off first since it is the sum of two transitions
2
 relaxation rates via dipolar coupling are:

3 c 3 c
W1A  
r 6 (1   A2 c2 ) r6
3 c 2 c NOE is dependent on the
W0  6  6 distance (1/r6) separating the
r ( A   X )  c )
2 2
r
two dipole coupled nuclei
12 c 12 c
W2  
r 6 (1  ( A   X ) 2 c2 ) r6
Important: the effect is time-averaged!

Extreme narrowing limit: 1/c >>o then o2c2 <<1)

One-Dimensional (1D) NMR Experiments
Nuclear Overhauser Effect (NOE)
Dependence of NOE on c
• NOE can be positive, zero or negative depending on c  MW

Zero NOE

positive NOE

negative NOE

Small molecules Increasing MW Biomolecules, polymers

Decreasing c
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   lower 
c max
 NOE builds up by dipole-dipole relaxation

– in small molecules, T
1DD > 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)
13
C 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

1
H 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 CH point down (negative)
3
• Maximize sensitivity by complete decoupling and NOE, but maintain spin
system information.

d1 = recycle delay 90o 180o

for relaxation

d2 = 1/J1H-13C
One-Dimensional (1D) NMR Experiments
J Modulation (JMOD)

Aids in NMR Assignments

• Identifies the number of different spin systems 10
presents 4
3
• Chemical shifts identifies the types of 1
6
functional groups that are present. 8
7

9
2 5

6 8

1 2 4

3
7 9,10
 One-Dimensional (1D) NMR Experiments
J Modulation (JMOD) On resonance
180o decouple
(center of coupling pattern)
Remember Coupling constants are in Hz
(cycles per second) 13
C
• complete cycle is 360 o

• each spin moves relative to carrier 13

CH
(center of spin system) during d2 delay

•13C singlet:
on resonance doesn’t move during 1/J

•13CH doublet each spin distance from CH2

13

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 13
CH3
•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
 One-Dimensional (1D) NMR Experiments
J Modulation (JMOD) On resonance
180o decouple
(center of coupling pattern)
Phase of the Peaks Differ as a result of
the Different Spin Systems 13
C
• the 180o pulse and the second 1/J delay
allows for refocusing of chemicals shifts that
13
CH
differ from the carrier position
 rotation is actually dependent on +J

180o reverses direction and refocus

rotation due to 

• 1J13CH ~ 125-170 Hz
CH2
13
 use average J ~ 145 Hz

 problems with 13CH of alkynes J ~250

Hz  behaves like 13CH2

•Decoupler is turned on during second d2 and

acquisition to collapse spins to singlet and
gain NOE sensitivity
13
CH3

• If d2 set to 1/2J, only observe 13C

 difficult  average J  incomplete

cancellation and weak 13C signal

One-Dimensional (1D) NMR Experiments
INEPT
Polarization Transfer
• population difference between  and  states is proportional to 
 1 H population difference ~ 4x > 13C
 If this difference could be transferred from 1H to 13C, 13C S/N would

increase by a factor of 4.
 Lose of NOE effect

• polarization transfer > NOE effect

One-Dimensional (1D) NMR Experiments
INEPT
Polarization Transfer
• selective 180o on one 1H spin
 inverts the 1H  and  spin states
 13C population differences are now ±H instead of +C

 Repeat by inverting other 1H 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

d1 = recycle delay
for relaxation

d2 = 1/4J1H-13C

180o 90o
One-Dimensional (1D) NMR Experiments
INEPT

Separation in peaks
indicate triplet (J~145Hz)

INEPT Pascal Triangle

J 2J

-1:1 doublet -1:0:1 triplet -1:-1:1:1 quartet

13
CH 13
CH2 13
CH3
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
 during first d2 = 1/4J each spin moves 45o

 180o 1H refocusing pulse flips spins (would refocus after another 1/4J delay

 180o X pulse exchanges a and b 1H spins

– X attached to a are now attached to b and vice-versa

– direction of rotation is reversed
 During second d2, each spin moves another 45o and are aligned 180o to each other

 900 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

 0.25/J only 13CH observed

 0.375/J CH are anti-phase (negative)

2
• 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 J
XH 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
 45o pulse: CH, CH and CH are all positive
2 3
 90o pulse: only CH signal observed

 135o pulse CH and CH positive with CH being negative

3 2
•Addition and subtraction of DEPT-45, DEPT-90 and DEPT-135 can generate spectra that
only contains CH, CH2 or CH3 signals

90o 180o o

d1 = recycle delay
for relaxation

d2 = 1/2J1H-13C
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

W1 A
W1X DEPT
W2
 
W0
W1X W1A

Wo,W2: multiquantum,
forbidden transitions
multiple quantum vector does
not change during 
C 90o creates multiple
13

quantum coherence

180o pulse refocus Anti-phase component

chemical shifts (amplitude function of sin )

Last 1H pulse

Multiquantum component
(amplitude function of cos )
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
 observes quaternary 13C, 13CH, 13CH and 13CH
2 3
 quaternary 13C signals are stronger than in JMOD

 C/CH are opposite phase of CH/CH signals

2 3
• 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

 pointless to acquire simple 1H decoupled 13C spectrum

 replaces JMOD and APT

• Again, simple spin vector diagrams are insufficient to describe pulse sequence
 Creating multiple quantum coherence

90o 180o
d1 = recycle delay
for relaxation

d2 = 1/4J1H-13C

d3 = 5/8J1H-13C
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
 probability of two bonded atoms both being 13C is 1.17e-2%

• Experiment suppresses strong center peak to detect 13C satellites

Center peak
off-scale

C2 C4
13
C satellites C5
C1 C3
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

1
H 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

C spectrum
13
One-Dimensional (1D) NMR Experiments

Summary of Information Present in Some 1D Experiments