Use of NMR in Structure

Elucidation

Presented By:

Anuradha Verma

Research Scholar

Overview

• NMR is a sensitive, non-destructive method

for elucidating the structure of organic

molecules

• Information can be gained from the hydrogens

(proton NMR, the most common), carbons

(13C NMR) or other elements like 31P, 15N, 19F.

Making NMR work

• Not all protons absorb at the same field values

• Either magnetic field strength or radio frequency must be varied

• Frequency/field strength at which the proton a sor s tells so ethi g a out the proto ’s surroundings

In a magnetic field the states have different energies

Alignment with the magnetic field (called ) is

lower energy than against the magnetic field

(called ). How much lower it is, depends on the

strength of the magnetic field

• Energy difference linearly depends on field

strength

E

Bo

E = h x 300 MHz E = h x 500 MHz

7.05 T 11.75 T

proton spin state (lower energy)

proton spin state (higher energy)

Graphical relationship between

magnetic field (B o) and frequency (

for 1H NMR absorptions

at no magnetic field,there is no difference beteen- and - states.

0 T

NMR Signals

• The number of signals shows how many different

kinds of protons are present.

• The location of the signals shows how shielded or

deshielded the proton is.

• The intensity of the signal shows the number of

protons of that type.

• Signal splitting shows the number of protons on

adjacent atoms.

Chemical shift

• Protons in different environments absorb at

different field strengths (for the same frequency)

• Different environment = different electron density

around the H

Location of Signals

• More electronegative atoms

deshield more and give larger

shift values.

• Effect decreases with distance.

• Additional electronegative

atoms cause increase in

chemical shift.

Chapter 13

Typical Values

Aromatic Protons, 7-8

Vinyl Protons, 5-6

Acetylenic Protons, 2.5

O-H and N-H Signals

• Chemical shift depends on concentration.

• Hydrogen bonding in concentrated solutions deshield

the protons, so signal is around 3.5 for N-H and 4.5

for O-H.

• Proton exchange between the molecules broaden the

peak.

Hydroxyl Proton

• Ultrapure samples of

ethanol show splitting.

• Ethanol with a small

amount of acidic or basic

impurities will not show

splitting.

N-H Proton

• Moderate rate of exchange.

• Peak may be broad.

Identifying the O-H or N-H Peak

• Chemical shift will depend on concentration and solvent.

• To verify that a particular peak is due to O-H or N-H, shake the sample with D2O.

• Deuterium will exchange with the O-H or N-H protons.

• On a second NMR spectrum the peak will be absent, or much less intense.

Carboxylic Acid Proton, 10+

Intensity of Signals

• The area under each peak is proportional to

the number of protons.

• Shown by integral trace.

Spin-Spin Splitting

• Nonequivalent protons on adjacent carbons have

magnetic fields that may align with or oppose the

external field.

• This magnetic coupling causes the proton to absorb

slightly downfield when the external field is reinforced

and slightly upfield when the external field is opposed.

The N + 1 Rule

If a signal is split by N equivalent protons,

it is split into N + 1 peaks.

=>

Chapter 13

Doublet: 1 Adjacent Proton

=>

Chapter 13

Triplet: 2 Adjacent Protons

=>

Range of Magnetic Coupling

• Equivalent protons do not split each other.

• Protons bonded to the same carbon will split each other only if they are not equivalent.

• Protons on adjacent carbons normally will couple.

• Protons separated by four or more bonds will not couple.

Coupling Constants

• Distance between the peaks of multiplet

• Measured in Hz

• Not dependent on strength of the external field

• Multiplets with the same coupling constants may

come from adjacent groups of protons that split each

other.

Values for Coupling Constants

Complex Splitting

• Signals may be split by adjacent protons, different

from each other, with different coupling constants.

• Example:

Ha of styrene which is split by an adjacent H trans to it

(J = 17 Hz) and an adjacent H cis to it (J = 11 Hz).

C C

H

H

Ha

b

c

General Regions of Chemical Shifts

Aldehydic

Aromatic and heteroaromatic

Olefinic

-Disubstitutid aliphatic

-Monosubstituted aliphatic

Acetylenic

-Substituted aliphatic

Aliphatic alicyclic

0 1 3 4 5 6 10 2 7 8 9 = TMS

CH3-CH2-CH2-CH2-CH2-CH=CH-CH2-CH=CH-CH2-CH2-CH2-CH2-CH2-CH2-CH2-COOCH2

HOCH

HOCH2

cyclohexane

a singlet 12H

2,3-dimethyl-2-butene

C

CH3

C

H3C

H3C

CH3

a singlet 12H

benzene

a singlet 6H

p-xylene H3C CH3

a a b

a singlet 6H

b singlet 4H

tert-butyl bromide

C CH3H3C

Br

CH3 a singlet 9H

ethyl bromide

a b

CH3CH2-Br

a triplet 3H

b quartet 2H

1-bromopropane

a b c

CH3CH2CH2-Br

a triplet 3H

b complex 2H

c triplet 3H

isopropyl chloride

a b a

CH3CHCH3

Cl

a doublet 6H

b septet 1H

2-bromobutane

b d c a

CH3CHCH2CH3

Br

a triplet 3H

b doublet 3H

c complex 2H

d complex 1H

ethylbenzene

CH2CH3

c

b a

a triplet 3H

b quartet 2H

c ~singlet 5H

di-n-propylether

a b c c b a

CH3CH2CH2-O-CH2CH2CH3

a triplet 6H

b complex 4H

c triplet 4H

1-propanol

a b d c

CH3CH2CH2-OH

a triplet 3H

b complex 2H

c singlet 1H

d triplet 2H

13C ~ 1.1% of carbons

1) number of signals: how many different types of carbons

2) chemical shift: hybridization of carbon sp, sp2, sp3

13C – NMR

2-bromobutane

a c d b

CH3CH2CHCH3

Br

•The magnetic nucleus may assume any one of ( 2 I + 1)

orientations with respect to the directions of the applied

magnetic field.

•Therefore, a proton (1/2) will be able to assume only one

of two possible orientations that correspond to energy

levels of + or - H in an applied magnetic field, where H

is the strength of the external magnetic field.

Summary

Summary

•If proper v is introduced, the Wo will be resonance with

the properly applied radio frequency and the proton will

absorb the applied frequency and will be raised to the

high energy spin state.

•Even though the external magnetic field strength (Ho)

applied to the molecule is the same, the actual magnetic

field strength exerted to the protons of the molecule are

different if the protons are in the different electronic

chemical environment.

Structure Determination of Biological

Molecules

Structure Determination by NMR

• Biological molecules such as proteins and nucleic acids can be large and complex. They can easily exceed 2000 atoms.

• Knowing their structure is critical in understanding the relationship between structure and function.

• X-ray crystallography is an excellent method to determine detailed 3D structures of even some of the largest biological molecules.

• However, it has some significant difficulties. Getting crystals and the obtained structure may not be biologically relevant.

• NMR can be used to determine 3D structure and dynamics in solution!

It’s li itatio is ole ular size. Ho e er, this is ha gi g.

• Large molecules with numerous atoms nuclear magnetic moment

does not permit the determination of these fundamental parameters

easily.

• Some 1D spectra are far too complex for interpretation because

signals overlap heavily.

• e.g. cholesterols, protein spectra

How 2D NMR is useful?

Nonequivalent proton groups can have nearly the same chemical shift and/or complex splitting patterns making 1D NMR spectra complicated even for relatively simple molecules.

The introduction of additional spectral dimensions simplifies the spectra and provides more information.

Two-dimensional (2D) NMR techniques can be used to solve such sophisticated

structural problems.

2-D spectra simplify the complexity arising from overlapping of peaks.

Simplification of NMR spectra makes their interpretation easier and sometimes the only way possible.

The interaction of nuclear spins (1H with 1H, 1H with 13C, etc.) are plotted in two dimensions

• In 2-D spectra the intensity is plotted as a

function of two frequencies, usually represented

as F1 and F2. F1 and F2 are Fourier transformed

frequency axis from a time domain signal.

H-H Correlation Spectroscopy (COSY)

• In a COSY experiment, the chemical shift range

of the proton spectrum is plotted on both axis.

• COSY spectrum of a molecule containing just one

type of protons HX.

• COSY spectrum of a hypothetical molecule

containing just two protons, HA and HX, which are

not coupled

• COSY spectrum of a hypothetical molecule containing just two types

of protons, HA and HX, which are coupled

• Signals on the diagonal divides the spectrum in two equal halves.

Signals symmetrical to the diagonal called cross signals (peaks).

• The cross signals originate from nuclei that exchanged magnetization

during the mixing time. They indicate an interaction of these two

nuclei. The cross signals contain the information of 2D NMR spectra.

• If there had been no coupling, their magnetizations would not have given rise to off-diagonal peaks.

• COSY spectrum shows which pairs in a molecule are oupled (thro’ ond oupling, hen e connectivity).

• From a single COSY spectrum it is possible to trace out the whole coupling network in the molecule.

31P - NMR

• 31P-NMR allowed the measurement of the

intracellular pH of the muscle, resting or

fatigued, through the shift of the frequency of

the Pi peak

• The major phosphate metabolites of muscle are:

ATP, PCr, Pi. ATP and PCr occur at high

concentrations in normal resting muscle,

whereas the appearance of Pi indicates fatigue.

31P-Spectroscopy of Heart Muscle

• 31P spectrum of beating rat heart shows the

Pi, PCr, and ATP resonances

Thank You