use of nmr in structure ellucidation

basics, principle, working, peaks, chemical shift

1. Use of NMR in Structure
Elucidation
Presented By:
Anuradha Verma
Research Scholar

2. 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
(
13
C NMR) or other elements like
31
P,
15
N,
19
F.

3. 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
asors tells souethivg aout the protov’s
surroundings

4. 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

5. •Energy difference linearly depends on field
strength

E
B
o
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
1
H NMR absorptions
at no magnetic field,
there is no difference beteen
- and - states.
0 T

6. 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.

7. Chemical shift
•Protons in different environments absorb at
different field strengths (for the same frequency)
•Different environment = different electron density
around the H

8. 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.

9. Chapter 13
Typical Values

10. Aromatic Protons, 7-8

11. Vinyl Protons, 5-6

12. Acetylenic Protons, 2.5

13. 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.

14. Hydroxyl Proton
•Ultrapure samples of
ethanol show splitting.
•Ethanol with a small
amount of acidic or basic
impurities will not show
splitting.

15. N-H Proton
•Moderate rate of exchange.
•Peak may be broad.

16. 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 D
2O.
•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.

17. Carboxylic Acid Proton, 10+

18. Intensity of Signals
•The area under each peak is proportional to
the number of protons.
•Shown by integral trace.

19. 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.

20. The N + 1 Rule
If a signal is split by N equivalent protons,
it is split into N + 1 peaks.
=>

21. Chapter 13
Doublet: 1 Adjacent Proton
=>

22. Chapter 13
Triplet: 2 Adjacent Protons
=>

23. 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.

24. 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.

25. Values for Coupling Constants

26. Complex Splitting
•Signals may be split by adjacent protons, different
from each other, with different coupling constants.
•Example:
H
a
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
H
a
b
c

27. 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
CH
3-CH
2-CH
2-CH
2-CH
2-CH=CH-CH
2-CH=CH-CH
2-CH
2-CH
2-CH
2-CH
2-CH
2-CH
2-COOCH
2
HOCH
HOCH
2

28. cyclohexane
a singlet 12H

29. 2,3-dimethyl-2-butene
C
CH
3
C
H
3C
H
3C
CH
3
a singlet 12H

30. benzene
a singlet 6H

31. p-xylene
H
3C CH
3
a
a
b
a singlet 6H
b singlet 4H

32. tert-butyl bromide
C CH
3H
3C
Br
CH
3 a singlet 9H

33. ethyl bromide
a b
CH
3CH
2-Br

a triplet 3H
b quartet 2H

34. 1-bromopropane
a b c
CH
3CH
2CH
2-Br

a triplet 3H
b complex 2H
c triplet 3H

35. isopropyl chloride
a b a
CH
3CHCH
3
Cl

a doublet 6H
b septet 1H

36. 2-bromobutane
b d c a
CH
3CHCH
2CH
3
Br

a triplet 3H
b doublet 3H
c complex 2H
d complex 1H

37. ethylbenzene
CH
2CH
3
c
b a
a triplet 3H
b quartet 2H
c ~singlet 5H

38. di-n-propylether
a b c c b a
CH
3CH
2CH
2-O-CH
2CH
2CH
3

a triplet 6H
b complex 4H
c triplet 4H

39. 1-propanol
a b d c
CH
3CH
2CH
2-OH

a triplet 3H
b complex 2H
c singlet 1H
d triplet 2H

40. 13
C ~ 1.1% of carbons

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

2)chemical shift: hybridization of carbon sp, sp
2
, sp
3

13
C – NMR

41. 2-bromobutane

a c d b
CH
3CH
2CHCH
3
Br

42. •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

43. 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.

44. Structure Determination of Biological
Molecules

45. 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 liuitatiov is uoleular size. Ho?e?er, this is havgivg.

46. •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

47. 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 (
1
H with
1
H,
1
H with
13
C, etc.) are plotted in two
dimensions

48. •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.

49. H-H Correlation Spectroscopy (COSY)
•In a COSY experiment, the chemical shift range
of the proton spectrum is plotted on both axis.

50. •COSY spectrum of a molecule containing just one
type of protons H
X.



•COSY spectrum of a hypothetical molecule
containing just two protons, H
A and H
X, which are
not coupled

51. •COSY spectrum of a hypothetical molecule containing just two types
of protons, H
A and H
X, 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.

52. •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, hene
connectivity).
•From a single COSY spectrum it is possible to
trace out the whole coupling network in the
molecule.

53. 31
P - NMR

54. •
31
P-NMR allowed the measurement of the
intracellular pH of the muscle, resting or
fatigued, through the shift of the frequency of
the P
i peak
•The major phosphate metabolites of muscle are:
ATP, PCr, P
i. ATP and PCr occur at high
concentrations in normal resting muscle,
whereas the appearance of P
i indicates fatigue.

55. 31
P-Spectroscopy of Heart Muscle
•
31
P spectrum of beating rat heart shows the
P
i, PCr, and ATP resonances

56. Thank You