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‘Introduction
¢-Fundamental principles and theory -Coupling co nstant
¢Instrumentation -2D-NMR

‘Solvents -NOE

¢Chemical shift -NOESY

¢Factors affecting chemical shift -COSY

‘Interpretation of proton -Applications

NMR spectra

Proton chemical shifts

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Nuclear Magnetic Resonant

technique which is based on the absorption of ele

in the radio frequency region 4 to 900 MHz by nuclei of the

atoms.

Proton Nuclear magnetic resonance spectroscopy is one of the

most powerful tools for elucidating the number of hydrogen or proton

in the compound.

It is used to study a wide variety of nuclei:

@ 1H 15N

+195 OF
13C 31

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Spin quantum number
the atomic and mass number of the nucle

i Atomic Atomic | Examples
Mass Number

Half- Odd Odd 1H (1/2)
integer NMR active

Half- Odd Even 13C (1/2)
integer
Integer Even Odd 2H (1)

zero Even Even 12C (0) Not NMR
active

Elements with either odd mass or odd atomic
number have the property of nuclear “spin”.

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possible orientations calculated by”

Hydrogen has spin quantum number I=1/:
possible orientation is (2*1/2+1=2) two +1/2 and -1/2.

Applied magnetic field
Energy +

etree

Spin state J=-1/2 (B) Precessional .~

n n dee Oe
Spinning
nucleus

ser tmen..

Spin state J =+1/2 (a)
meee eo

Field strength By :

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J a”
The theory behind NMR com 7

from the spin of a nucleus and it QQ
o” Or

generates a magnetic field. Without an P
On

external applied magnetic field, the
No external magnetic field

nuclear spins are random in directions.

But when an external magnetic
o 9 9

field(B,), is present the nuclei align é

themselves either with or against the } > ?
?

field of the external magnet. ?

With external magnetic field
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External magnetic field

 

applied, an energy transfer (AE)
possible between ground state to
excited state. +4(ore) -4(or 6)

when the spin returns to its Spin Energy States

ground state level, the absorbed

radiofrequency energy is emitted at A he,
y -spin state

the same frequency level. UL’
PS AE

The emitted radiofrequency ‘2 <
c th

signal that give the NMR spectrum of Pe s
” Q-spin state

the concerned nucleus. ¥
Applied Magnetic

aes Field Strength —e
Increases

Bo

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The em itted radio fre
to the strength of the applied field.

B = External magnetic field experienced by proton

y = Magnetogyric ratio ( The ratio between the nuclear

magnetic moment and angular moment)

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RS.

Int + shielded
ntensity ae

d eshi elded nuclei nuclei

TMS

downfield <— upfield

Frequency

5

The NMR spectrum is a plot of intensity of NVR

signals VS magnetic field (frequency) in reference to TMS

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1. Sample holder :
2. Permanent magnet 5. Radio fi

3. Magnetic coils 6. Radio frequen

7.Read out systems

 

radiofrequency radiofrequency |

transmitter amplifier | : detector |

audio |

amptitier | ha Fu—]

 

oscilloscope
and /or

 

recorder

sweep
generator

 

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1.Sample holder :- Glas s tube
in diameter

2.Permanent magnet _:- It provides homogeneous magnetic

field at 60-100 MHZ

3.Magnetic coils :- These coils induce magnetic field

when current flows through them.

4.Sweep generator :- To produce the equal amount of

magnetic field pass through the sample

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5-Radio frequency _ :- A radio transmitter

transmitter that produces a short powerful

pulse of radio waves

6.Radiofrequency —:- A radio receiver coil that detects Receiver

radio frequencies emitted as

nuclei relax to a lower energy level

7.Readout system — :- A computer that analyses

and record the data

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The fol lowing solvents are normally used in wh
hydrogen replaced by deuterium.

CCl4 _ – carbon tetrachloride

CS2__- carbon disulfide

CDClg – Deuteriochloroform

C6D6 – Hexa deuteriobenzene

D,O – Deuterium oxide

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A chemical shift is defined as the differe

per million (ppm) between the resonance frequency of the

observed proton and tetramethylsilane (TMS) hydrogens.

TMS is the most common reference compound in NMR,

it is set at6=0 ppm

Chennical si, 3 = frequency of signal – frequency of reference aT

spectrometer frequency
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High electron der
nucleus from the external magnetic f

upfield in the NMR spectrum

Deshielding of protons:-

Lower electron density around a nucleus deshields

the nucleus from the external magnetic field and the

signals are downfield in the NMR spectrum

I : | I

10 = 8 7 6 >

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=

—{

4

4

+

4

4

—{

=~

4

4

 

lectronegative groups

¢ Magnetic anisotropy of m-systems
¢ Hydrogen bonding
Electronegative groups:-

Electronegative groups attached to the C-H system

decrease the electron density around the protons, and there is

less shielding (7.e.deshielding) and chemical shift increases

CH,I 2.16

CH,Br 2.65

CH,Cl 3.10

CHF 4.26

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The wo rd “anisotropic !
anisotropy means that there is a “non-uni

> Electrons in 1 systems (e.g. aromatics, alk enes, alky nes, ¢
etc.) interact with the applied field which induces a magnetic field that

causes the anisotropy.

> It causes both shielding and deshielding of protons.

> Example:-Benzene

Hydrogen bonding:-

> Protons that are involved in hydrogen bonding are typically change

the chemical shift values.

> The more hydrogen bonding, the more proton is deshielded and

chemical shift value is higher. — wwwww.d.Duluolom Mixix..ccoomm Page 17

 

The most common for of NMR is based on the

hydrogen-1 (*H), nucleus or proton.

It can give information about the structure of any

molecule containing hydrogen atoms.

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Approximate Approximate Type af Ap proximate Approximate
chemical sWin chemical shift

Type of proton ¢ppen) (pem) carbon chemical shift (ppm) chemical shift (ppm)

(CHyS 0
4S-—8 (CH), Si Oa

—CH; 0.9

RCH 3-63
8.3 90-10

—ey— 4$=80
14

2S—4 R=CHy—R 60
S80

—CmC—CHy 1.7

24=4
0 ROHR 20-4) 165-178

—d_ ch,

4

R—C—R 3040) 168-178
41—i5

—CaeC—H

R=—O—CH, Variable. | 4-4
=C 6$-85 178-188

Variable, 2-5
R—C CH;

Alt Variable, 4-7

R—C-C—H

kk ee Variable, 10-12

0

— tnt; Variable. £-8

OTe wee oC ppmumae Doomed Oey wt cMEctal by oo Girton goatee teu 100=1 $0 190—20X)

CF 110-170 208-220
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H NMR Table

 

==

= O— cC—
fe)

BA SD

= (ByC1¢c — —

o (H)
I N —c-—@® |

ee pi ecas
6 Hy

Cease Mize See A

nine ee “@
yD Pind on Pol’y hat

feo ae E :

ppm,

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fy e

T T T T T T

8 ¥ 6 5 4 3 2 1 0
6(ppm)

‘H spectrum of Ethanol:-
3 types of proton

CH, CH, OH
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|

fF oe” § 7 he , * | * 0
PPM

cal shift scale 6.5-8.0 ppm.
– Aromatic Hydrogen shows peak in the chemi

of protons and it shows
– From the above spectrum Benzene has same type

single peak at 7.26
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Number of signals – Indicates how many “diffe

protons are present.

Position of signals – Indicates something about (chemicalshift)

magnetic (electronic) environment of protons

Relative intensity of – Proportional to number of protons present

signals

Splitting of signals – Indicates the number of nearby nuclei

(spin spin coupling) usually protons

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: The multiplicity of signal is cale

A?° ° This is one of the rule to predict the splitting of proton

signals. This is considered by the nearby hydrogen nuclei.

Therefore, n= Number of protons in nearby nuclei

> Zero H atom as neighbour n+1=0+1=1(singlet)

> One H atom as neighbour n+1=1+1 = 2(doublet)

> Two H atom as neighbour n+1=2+1 =3(triplet)

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The interaction b etween
nuclei in a molecule may cause the sphitt
spectrum.This is known as spin-spin coupling OF ST

The splitting pattern is related to the number of
equivalent H-atom at the nearby nuclei.

pin-Spin Splitting patterns
mg acetate

S

CH, 1. Singlet. 3 equivalent
me protons. Not coupled to any

es hy en<-0 ppm neighboring protons.

/ 3 2. Quartet. 2 equivalent
Ps protons. Split (1°3:3°1)

3 because coupled to the 3
‘Hs at the 2 position

6=4.1 ppm ppm | 3. Triplet. 3 equivalent
protons. Split (1.2.1)

2 because coupled to the 2
1Hs at the 3 position.

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Wi

 

* Chemically equi valent protons do no ts
* Only nonequivalent protons couple.

in t ; in 1 H, couples with H..
H—C—C—C—C—H H, and H, do not couple because they are equivalent.

| | | | H. and H, do not couple because they are equivalent.

* Protons on adjacent carbons normally will couple.

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

a a i H, can couple with H,
H .—c—c—c H, can couple with H,
a

H, cannot couple with H,

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The distance between the

 

a measure of the splitting effect known as cot DTS “CO

° It is denoted by symbol J, expressed in Hz.

. Coupling constants are a measure of the effectiveness

of spin-spin coupling and very useful in ‘H NMR of complex

structures.

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cs of 2D NM R Experiment:-
Basis: Interaction of nuclear spins (‘H with 1H, !
dimensions

* Normal spectra( 1D NMR) are plots of intensity Vs frequency.

* In 2D NMR intensity is plotted as function of two frequencies called f, & f,

¢ In general, 2D’s can be divided into two types,

Homonuclear

Heteronuclear

¢ Each type can provide either through-bond

(COSY-type) or through space (NOESY-type)

coupling information

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¢ Correlates one spectrum
with another

* May be the same or
different nuclei
— 1H-1H
7 THA13¢C —

— 130-13C 1-15) |
– May correlate differently :

* COSY — through bond os
* TOCSY — extended system LOUD eUbUG amub al in
¢ NOESY — through space

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at

 

2-D NMR -Signal is recorded as a function |
variables, tz and t2.

evoltue tion Fring} detection

1

Pulse Sequence

¢Rf pulses are generally applied during the preparation and
mixing periods.

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_ Applications of 2D NMR:-
“¢ Simplifies analysis of more complex or air

cases such as proteins.

¢* Obtain structural information not accessible by one-

dimensional NMR methods.

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Techniques in clude:
¢ Correlation Spectroscopy (COSY)

¢ Heteronuclear Correlation Spectroscopy (HETCOR)

¢ Heteronuclear Multiple-Quantum Coherence (HMQC)

¢ Nuclear Overhauser Effect Spectroscopy (NOESY)

¢ Incredible Natural Abundance Double Quantum Transfer

Experiment (INADEQUATE)

¢ Many others

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COrrelationSpe ctroscopY(COSY) —Scalé
» Identifies all coupled spins systems.

Nuclear OverhauserEffect SpectroscopY(NOESY) —Dipolar

Coupling

»Identifies neighboring spin systems (<5 A)

»Identifies chemical exchange.

Heteronuclear Multiple/Single Quantum Correlation

(HMQC/HSQC) —Scalar Coupling

» Identifies coupling between heteronuclei(C-H, N-H)

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*OSY: Homonuclear correla t
between protons that are couple

TOCSY: Total Correlation Spectroscopy- Uses a spin-lock for

coherence transfer. During the spin-lock all protons of

a coupled system become “strongly coupled,” leading to

cross peaks between all resonances of a coupled system.

HETCOR: Heteronuclear correlation, usually between “H

and ‘°C resonances mediated by J¢.;;

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~NOESY, R OESY: Proto n-pro
dipolar coupling (NOE effect). Correl:

protons that are close in space. This is the single most

powerful NMR technique for determining the 3-

dimensional structure of molecules from

conformations of small molecules to the 3-dimensional

structure of small proteins.

HOESY: Heteronuclear Overhauser Effect Spectroscopy.

Correlation between protons and heteronuclei that are

close in space.

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*Correlation Spectroscopy
«Commonly used for regiochemical assignment

*Cross-peaks appear if spin coupling is present

*Protons that are separated by 2 or 3 bonds are usually detected

 

= _ – too

ae if
= – s ba |
2 && ? arf _

— Be) Bde a4 _
240 700 1= 795

COSY spectrum of 3-heptanone a . age 36
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NOE Caused by di polar coupling
° The local field at one nucleus is affected by the presence
of another nucleus. The result is a mutual modulation of
resonance frequencies.

6 6

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The intensity of the interaction is a fur
distance between the nuclei according to the following aque

I – intensity
I=A(1/r°) —A-scaling constant

r – internuclear distance
r

(H) —_> (H) Arrows denote cross relaxation pathways
r,. – distance between protons 1 and 2

i r,. – distance between protons 2 and 3
ri3 ip =o

y 23
(1) The NOE provides a link between an

experimentally measurable quantity, I, and
internuclear distance.
NOE is only observed up to 5 A.
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Propyl Benzoate NOESY
Nuclear Overhauser
Effect Spectroscopy Lal LL

Shows correlations to
protons that are nearby le
in space to about 4
angstroms (most J
coupling is filtered out).

Interpret by
Noting which protons

are close to one another
(very useful for
stereochemical
analysis).

Protons that are geminal
and vicinal will still

 

appear because they
— —~?d – – 8

are close.
poorer

85 80 75 7.0 65 60 55 50 45 40 35 30 25 20 4. 5 10 ppm

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Y NMR is used in biolo gy to study the Biofluids, Cells, –
organs and biomacromolecules such as Nucleic acids(DNA,

RNA), carbohydrates Proteins and peptides. And also

Labeling studies in biochemistry.

vY NMR is used in physics and physical chemistry to study High

pressure Diffusion ,Liquid crystals ,liquid Crystal solutions ,

Membranes, Rigid solids.

Y NMR is used in food science.
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VY NMRis used in pharmaceutical science to Stud

Pharmaceuticals and Drug metabolism.

Y NMR is used in chemistry to ;
° Determine the Enantiomeric purity.
° Elucidate Chemical structure of organic and

inorganic compounds.
° Macromolecules —ligand interaction.

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applica tio ‘H widely used for structure elucidation.
Inorganic solids- In organic compounds are investigated by

solid state 1H-NMR.eg CaSO4-H20.

Organic solids- Solid-state ‘H NMR constitutes a powerful

approach to investigate the hydrogen-bonding and ionization

states of small organic compounds.

¢ Direct correlation with hydrogen-bonding lengths could

be demonstrated, e.g. for amino acid carboxyl groups.

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eptides and proteins
Clinical and scientific research
In vivo NMR studies-
° concerned with ‘H NMR

spectroscopy of human brain.
° Many studies are concerned with altered levels

of metabolites in various brain diseases.
To determine the spatial distribution of any
given metabolite detected spectroscopically IS
(image selected in vivo spectroscopy).

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MRI is specia list application of mult
Fourier transformation NMR

v Anatomical imaging.

Y Measuring physiological

functions

¥ Flow measurements and

angiography.

v Tissue perfusion studies.

Y Tumors

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> Organic spectroscopy by William Kemp

> Instrumental methods of chemical analysis by Chatwal

> Instrumental methods of analysis by Willard

> Wikipedia.org

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SZ Op”
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