NMR Spectroscopy - SINAPSE · NMR 1-Dimension 1H NMR spectroscopy is ~50year old Over that time NMR has moved on considerable eg multi-dimension, multi-nuclei and very high field

NMR SpectroscopyWilliam Holmes

General Spectroscopy

“Spectroscopy is the study of the nature of energy levels of

matter, and the transitions induced between the by EM

radiation.”

•Energy levels quantised ie have descrete values (electron/nuclear,

nuclear/electron magnetic moments, vibrational, rotational etc).

•Interaction with Electromagnetic (EM) radiation.

•Emmision, Absorption, scattering or resonant.

•Selection rules

MRI, NMR

(energy levels of nuclear

magnetic moments)

Electron Spin Resonance (ESR, EPR)

IR (atomic bond vibrations)

Ultra Violet/visible (electron

transitions)

Xray Cystallography (scattering)

Types of Spectroscopy

Certain nuclei possess a nuclear magnetic moment (eg 1H, 13C, 19F,…)

Nucleus

Where is the signal from?

Patient Molecules 1H Atom 1H Nucleus Compass Needle

“Nuclear Magnetic

moment ”Electron

“spin angular

Momentum”

N

S

Polarisation using Strong Magnet

At atomic level quantum mechanics applies to nuclear magnetic moments (“spins”).

It says only TWO eigenstates are possible, the “spin up” and “spin down” state.

Ener

gy

No net Magnetisation M

M= N↑ - N↓ = 0

Compass Needle

N

S

No magnetic field



It says only TWO states are possible, the “spin up” and “spin down” state.

Ener

gy

magnetic

field, B S

N

Low energy

High energy

polarisation creates a Net Magnetisation M

This is the origin of MRI signal

M

M= N↑ - N↓ ≈ 6 per million

Compass Needle

N

S



It says only TWO states are possible, the “spin up” and “spin down” state.

Ener

gy

Compass Needle

N

S

∆E = hν

Frequency, ν

RF radiation of the precise frequency can drive transitions,

1. leading to an adsorption of energy.

2. Creation of transverse magnetisation

Signal Detection• Following pulse of RF radiation, Magnetisation M now precessing in xy plane

• Induces an electric current in the receiver coil. (Analogous to electric generator)

• Frequency of signal same as frequency of precession of M.

MRI Signal

y

x

B0

z

Φ

M

Receiver coil

Time (s)

∝precession

frequency, ωmagnetic field

strength B0

0Bγω =

High Resolution Liquid State Nuclear Magnetic Resonance

1. Chemical Shift

2. J coupling

3. Assigning spectra

4. Examples of metabolites

Chemical Shift

The resonant frequency depends on the main magnetic field B0

BUT, electrons can slightly alter the effective magnetic field

“seen” by a nucleus.

0Bγω =

Within a molecule 1H nuclei have,

• Different “electron environments”

• experience effective magnetic field, Beff

• Resonate at different frequencies

CH3—CH2 — OH

Chemical Shift

Nucleus experiences lower effective magnetic field

Beff = B0 - σB0 = (1- σ)B0

ω = γ(1-σ)B0

Magnetic

field

Electrons

induced to

circulate

Generating a small

additional magnetic

field opposing the

main field.

σ Chemical shift sheilding constant

C C

HA O

F HB

O strongly “electronegative”, ie

pulls electron density towards it.

Nucleus is Well shielded,

resonates at lower frequency

Nucleus is less Well shielded,

resonates at higher frequency

Chemical shift depends on “Function groups”

Example

Fourier

transform

freq

Intensity

C C

HA O

CH3HB

O is electronegative, ie pulls

electron density towards it.

Nucleus is Well shielded,

resonates at lower frequency

Nucleus is less Well shielded,

resonates at higher frequency

Example

ν0δ =

ν - ν0

Chemical Shift- the “ppm scale”

Resonance frequencies vary with magnetic field.

Therefore, Chemical Shift present with δ scale , unit ppm (parts per million)

This allows comparision between measurements made at

different magnetic fields (eg 1.5T and 3T).

Reference Standards

Organic Chemistry - Tetramethylsilane (TMS) define 0 ppm

In-vivo - internal reference e.g. N-acetyl aspartate to define 2.2ppm

CH3

Si

CH3

CH3

CH3

Chemical shift δ (ppm)

Chemical Shift

Effect of neighbouring functional groups

H nucleus become less shielded

Resonant at higher frequency


Chemical Shift


CSi

C

C

CH

H

H

H

HHH

HH

H

HH

H nuclei are well shielded

Resonant at lower frequency

TMS

C O

H


Chemical Shift


H nuclei LESS shielded

Resonant at higher frequencyCarbonyl group


Chemical Shift


C

H

H

For CH2 wide range

of ppm

Methylene group? ?

Remember

Number of 1H is proportional to AREA of peak(s) (NOT height!)

J Coupling (spin-spin Coupling)

The magnetic dipoles of 1H can interact via electrons.

C C

HA HB

Cl R

HA

Chemical ShiftB

ν

Resonance of HA depends on state of HBElectron

density

(Probability)



C C

HA HB

Cl R

HA

Chemical Shift

Chemical shift +

J coupling

Bα

ν

να



C C

HA HB

Cl R

HA

Chemical Shift

Chemical shift +

J coupling

B βν

νβ



C C

HA HB

Cl R

HA

Chemical Shift

Chemical shift +

J coupling

B βν

ναβ

Overall, molecules chance are 50:50

whether HB is up (α) or down (β), so

see both

Intensity ratio 1 : 1

Called a “Doublet”

α



C C

HA HB

Cl R

HA

Chemical Shift

Chemical shift +

J coupling

J Coupling

Constant Hz

B βν

ναβ



C C

HA H

Cl H

BHA

Chemical Shiftν



C C

HA H

Cl H

Bα

α

HA

Chemical Shift

Chemical shift +

J coupling

ν

ναα



C C

HA H

Cl H

Bβ

β

HA

Chemical Shift

Chemical shift +

J coupling

ν

νββ



C C

HA H

Cl H

Bα

HA

Chemical Shift

Chemical shift +

J coupling

ν

νβ αβ



C C

HA H

Cl H

Bβ

HA

Chemical Shift

Chemical shift +

J coupling

ν

να

βα



C C

HA H

Cl H

Bβ

HA

Chemical Shift

Chemical shift +

J coupling

ν

να

Intensity ratio 1 : 2 : 1

Called a “Triplet”

ββ αββα

αα

J

0 1 1 Singlet

1 2 1 1 Doublet

2 3 1 2 1 Triplet

3 4 1 3 3 1 Quartet


“n” magnetically equivalent neighbouring 1H, leads to “n+1” lines in multiplet.

nn+1

lines Intensities Name

C C

HA H

Cl H

H

Pascal’s Triangle

Simple rules for predicting NMR spectra

1. Divide hydrogens 1H into magnetically equivalent groups.

2. Look at functional group, estimate Chemical Shift.

Example Pentanone CH3—CH2 —C —CH2 —CH3

O

These are two magnetically equivalent groups of H

• The CH3 hydrogens (~1.7ppm)

• The CH2 hydrogens (~3.9ppm)

These will have different chemical shifts, hence expect two peaks.

CH2 CH3

0 ppm1.7 ppm3.9 ppm

3. Overall intensity of group proportional to number of

Hydrogens.

CH2

CH3

0 ppm1.7 ppm3.9 ppm

6 x CH3 hydrogens

4x CH2 hydrogens

Hence, ratio of peaks is 6:4


O


4. J Splitting due to neighbouring 1H, ( n magnetically

equivalent), leads to n+1 lines in multiplet.

CH2

CH3

0 ppm1.7 ppm3.9 ppm

CH3 hydrogen has CH2 as neighbours (2 equivalent protons) .

This splits the CH3 peak into (2+1= 3) ie triplet

CH2 hydrogen has CH3 as neighbours (3 equivalent protons) .

This splits the CH3 peak into (3+1= 3) ie quartet


O


5. Intensity within multiplet obeys Pascals Triangle

CH2

CH3

0 ppm1.7 ppm3.9 ppm


O

1 Singlet

1 1 Doublet

1 2 1 Triplet

1 3 3 1 Quartet

Quartet 1 : 3 : 3 : 1 Triplet 1 : 2 : 1


Chemical shift (ppm)

CH3—CH2 —C —CH2 —CH3

O

CH2 Quartet CH3 Triplet

Example: Lactate

Evolution of J coupling

with increased echo time

Example: GABA

Evolution of J coupling

with increased echo time

Example: Glucose

Spectra is mixture of two

anomeric forms, α-glucose β-

glucose.

Point Resolved Spectroscopy

Three slice selective pulses ONLY excite the

central Voxel.

Water suppression (Vapor,Chess) water signal >> metabolites, have to suppress

water signal

Outer volume suppression (see later)

Navigator echo is magnetic field drifts during scans, the navigator echo can be used

as reference. Prevents broadening of spectra.

Outer Volume Suppression

Without, OVS

With, OVS

Used to remove fat signal that is OUTSIDE the voxel

1H Spectra of Rat Brian

Example of 2-D NMR spectra

21Tesla magnet,

Birmingham

University, UK

NMR

1-Dimension 1H NMR spectroscopy is ~50year old

Over that time NMR has moved on considerable eg multi-dimension,

multi-nuclei and very high field.

NMR is a hugely important technique in Chemistry.

Now being used to study protein structure/folding.

Documents

NMR Spectroscopy - SINAPSE · NMR 1-Dimension 1H NMR spectroscopy is ~50year old Over that time NMR has moved on considerable eg multi-dimension, multi-nuclei and very high field