Structure Elucidation through NMR Spectroscopy. Structure Elucidation through NMR... · Structure Elucidation through NMR Spectroscopy Dr. Amit Kumar Yadav Assistant Professor-Chemistry

Structure Elucidation through

NMR Spectroscopy

Dr. Amit Kumar Yadav

Assistant Professor-Chemistry

Maharana Pratap Govt. P.G. College, Hardoi

Introduction

NMR is the most powerful tool

available for organic structure

determination.

It is used to study a wide variety of

nuclei:

1H

13C

15N

19F

31P

Nuclear Spin

A nucleus with an odd atomic number or an odd

mass number has a nuclear spin.

The spinning charged nucleus generates a

magnetic field.

External Magnetic Field

When placed in an external field, spinning

protons act like bar magnets.

Two Energy States

• The magnetic fields of the spinning nuclei will align either with the external field, or against the field.

• A photon with the right amount of energy can be absorbed and cause the spinning proton to flip.

E and Magnet Strength

• Energy difference is proportional to the magnetic field strength.

E = h = h B0 2

• Gyromagnetic ratio, , is a constant for each nucleus (26,753 s-1gauss-1 for H).

• In a 14,092 gauss field, a 60 MHz photon is required to flip a proton.

• Low energy, radio frequency.

Magnetic Shielding

• If all protons absorbed the same amount of energy in a given magnetic field, not much information could be obtained.

• But protons are surrounded by electrons that shield them from the external field.

• Circulating electrons create an induced magnetic field that opposes the external magnetic field.

Shielded Protons

Magnetic field strength must be increased for a shielded

proton to flip at the same frequency.

The NMR Spectrometer

Tetramethylsilane

Si

CH3

CH3

CH3

H3C

• TMS is added to the sample.

• Since silicon is less electronegative than carbon, TMS protons are highly shielded. Signal defined as zero.

• Organic protons absorb downfield (to the left) of the TMS signal.

Chemical Shift

• Measured in parts per million.

• Ratio of shift downfield from TMS (Hz) to total

spectrometer frequency (Hz).

• Same value for 60, 100, 300 MHz or above machines.

• Symbolized as delta scale.

Electronegativity

o Electronegativity can be a guide to chemical

shift only up to a point where other effects

are not operating. Degree of shielding

depends on the density of the circulating

electrons on the particular nucleus which will

directly depend on the inductive effect of the

attached groups.

o More electronegative atoms deshield more

and give larger shift values.

o Effect decreases with distance.

o Additional electronegative atoms cause

increase in chemical shift.

Factors Influencing Chemical Shift

Van Der Waals Deshielding

o Steric hinderence causes electrostatic repulsion which will

tend to repel the electron surrounding the proton.

o The proton will be deshielded and appear at higher value.

(less than 1 ppm),

o Must be taken in to account while predicting chemical shifts of

overcrowded molecules like steroids, triterpenoids, alkaloids

etc.

Anisotropic effects

o This effect depends on the diamagnetic anisotropy, which

means that shielding and deshielding depend on the

orientation of the molecule with respect to the applied

magnetic field i.e the effects are paramagnetic in certain

directions around the clouds and diamagnetic in others

hence anisotropic as opposed to isotropic (operating equally

through space).

Aromatic Protons, 7-8 Vinylic Protons, 5-6

Acetylenic Protons, 2.5

Aldehydic Protons, 9-10

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.

All possibilities exist, so signal is split.

• If a signal is split by N equivalent protons, it will split into N + 1 peaks.

The N + 1 Rule

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 of coupling constant

Complex Splitting

C C

H

H

Ha

b

c

• 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).

• The most common coupling constant we’ll see is the three bond

coupling, or 3J:

• As with the 1J or 2J, the coupling arises from the interactions

between nuclei and electron spins. 1J and 3J will hold the same sign,

while 2J will have opposite sign.

• However, the overlap of electron and nuclear wavefunctions in the

case of 3J couplings will depend on the dihedral angle <φ> formed

between the CH vectors in the system.

• The magnitude of the 3J couplings will have a periodic variation

with the torsion anlge, something that was first observed by Martin

Karplus in the 1950’s.

The Karplus equation

The magnitude of the 3J couplings will have a periodic variation with the torsion anlge, something that was first observed by Martin Karplus in the 1950’s.

The relationship can be expressed as a cosine series:

A, B, and C are constants that depend on the topology of the bond (i.e., on the electronegativity of the substituents). Graphically, the Karplus equation looks like this:

A nice ‘feature’ of the Karplus equation is that we can estimate dihedral angles from 3J coupling constants. Thus, a variety of A, B, and C parameters have been determined for peptides, sugars, etc., etc.

Carbon-13

• 12C has no magnetic spin.

• 13C has a magnetic spin, but is only 1% of

the carbon in a sample.

• The gyromagnetic ratio of 13C is one-fourth

of that of 1H.

• Signals are weak, getting lost in noise.

• Hundreds of spectra are taken, averaged.

=>

1H and 13C Chemical Shifts

2D NMR Techniques

1H-1H COSY (scalar coupling i.e. δ δ correlation spectroscopy)

J-Resolved spectroscopy (HOMO 2DJ): one axis contains δ values which are

correlated to J values on the other axis.

COSY-45: Modification of COSY to reduce the intensity of the diagonal signals

with respect to the intensity of cross peaks. Possible overlaps can thus be

avoided.

DQF-COSY (Double quantum filter):

Provides better visualization of cross peaks nearer to diagonal.

This sequence preferentially attenuates the single quantum resonances of the

diagonal with respect to cross peaks and also suppresses the detection of

isolated protons such as those arising from solvent or isolated methyl groups

i.e. magnetization of singlet signals is suppressed.

TQF-COSY (triple quantum filter) :

All the spin systems that contain less than three or more mutually coupled

spins are eliminated by use of TQF.

Example of such system is; Hexopyranosides where H-5, H-6 and H-6' cross

peaks were eliminated in TQF-COSY spectrum. Rings containing equatorial

protons prevent coherence or polarization transfer fron H-1 to H-5 and H-6 in

RELAY experiments.

1. Homonuclear COSY (Correlation spectroscopy):

PS-COSY (Phase sensitive): The basis for achieving remote

connectivities. Crtoss peaks obtained at F-2 (horizontal axis) of one

and F-12 (vertical axis) of another proton not only showed coupling

between themselves (active coupling) but also with other protons

(passive coupling). Cross section through one peak parallel to f-2 shows

multiplet pattern of this resonance and vice-versa for F-1 plane.

J2,3, J3,4, J4,5ax are in the range 8-10 Hz for diaxial gluco. where as

for galacto confor. J4,5 is 2 Hz.

Similarily they can be useful for identification of anomeric

conformation in gluco, galacto or manno configurations.

O

OH

O

O

O

H

O

H

H

O

O

OH

O

O

O

H

O

H

H

O

1H NMR spectrum

O

OH

O

O

O

H

O

H

H

O

O

OH

O

O

O

H

O

H

H

O

13C NMR spectrum

DQF-COSY spectrum

O

OH

O

O

O

H

O

H

H

O

HETCOR (13C-1H Heteronuclear Correlation Spectroscopy) Each cross

peak arises from connectivity between a 13C nucleus and its directly bonded

protons having the coordinates (δC, δH).

(It must be mentioned that in such experiments only 1 % of the protons

which are coupled to 13C are actually detected (due to less abundance of

13C nuclei) as compared to 1H detected experiments.)

HSQC (Heteronuclear single quantum coherence)

HMQC (Heteronuclear multiple quantum coherence)

HSQC and HMQC both are 1H detected experiments and provide one bond

correlation with high resolution in 13C domain.

COLOC (Correlation via Long range Coupling):

13C detected experiment where long range 13C, 1H couplings are observed.

HMBC (Heteronuclear multiple-bond correlations): 1H detected

experiments superior to COLOC by its reliability in long range correlations.

Long range three bond correlations appear in the spectrum which facilitates

in determining the remote connectivities in structural framework.

2. Heteronuclear Correlation spectroscopy

Gradient HSQC spectrum of 2a

O

OH

O

O

O

H

O

H

H

O

Gradient HMBC spectrum

O

OH

O

O

O

H

O

H

H

O

These experiments are based on polarization transfer phenomenon. In this

approach whole coupling network can be translated.

HOHAHA (Homonuclear Hartmann-Hann Spectroscopy) or TOCSY (Total

Correlation Spectroscopy):

The most useful method of relay of coherence along the chain of spins is the

isotopic mixing experiments in which the net magnetization is transferred under

spin locking.

Helpful in determining the J-network (group of protons serially linked via 1H

1H J (scalar) couplings.

Requirement of the experiment is presence of at least one well resolved signal of

the J-network (as anomeric proton in the case of oligosaccharides)

During spinlock or mixing time magnetization transfer takes place and short

mixing time (20-50 ms) leads primarily cross peaks of strongly coupled protons

while longer mixing time (100-300 ms) allows magnetization transfer to remote

protons of the spin system.

3. RELAY Experiments

OR1O

R1O

OOR1

OR1

O

R1O

OOR1

OR1

O

OR2

R1O

OR1

R2O

R1= Bn, R2R2= Benzylidene

1 2

1'2'

1''2''

1D TOCSY spectrum

ppm

1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5 ppm

1

2

3

4

5

6

7

8

TOCSY spectrum

O

N

CH3

NH2

O

CH3

Thr

O O

Val

OxoproSar

O

N-MeVal

Thr

Val

ProSar

O

N-MeVal

4. Dipolar Couplings:

NOESY (Nuclear Overhauser effect spectroscopy):

NOE connectivities are often observed between signals which are close oriented

in the three dimensional space.

The presence of inter-residue NOE from the anomeric ptoton of a particular

sugar residue to protons of the other sugar residues in case of oligosaccharides

or to non sugar residues in case of glycosides, defines the glycosidic linkage

between the two residues.

It depends upon spatial proximity of the protons.

ROESY (NOE in Rotating frame):

Due to problems with NOE measurements at medium field strength in NOESY,

ROESY can produce reliable NOE cross peaks not obtained from NOESY.

ROESY spectrum

O

OH

O

O

O

H

O

H

H

O

H-8

H-5

H-6

H-9

H-15

CH3-20

CH3-19

CH3-18

CH3-17

C-12

2a

O

OH

O

O

O

H

O

H

H

O

H-5

H-6

H-8

H-9

CH3-19

CH3-20

CH3-17

C-12

CH3-18

H-15

H-7

1a

6-COCH3

ROESY spectrum of the epimer

HR MAS NMR

High-resolution magic-angle spinning (HRMAS) probes allow NMR spectra to be

collected on a wide range of heterogeneous samples primarily because they average

the inhomogeneity created by magnetic susceptibility differences.

Tissue homogenates, soil samples, whole cells, solid phase organic synthesis and the

study of chromatographic stationary phases are a few applications where HRMAS

NMR probes have found widespread usage.

One reported application that has received very little attention is the use of HRMAS

NMR to characterize compounds directly from separated thin-layer

chromatography (TLC) spots.

In a preliminary report, Wilson et al. demonstrated that NMR spectra could be

obtained for model compounds separated by reverse-phase TLC (RPTLC) simply by

removing the separated spots from the RPTLC plate, transferring the dry powder

containing the adsorbed analyte to an HRMAS sample rotor, and forming a slurry

with D2O.

Wilson ID, Spraul M, Humpfer E. J. Planar Chromatogr. – Mod. TLC 1997; 10: 217.