special one dimensional, and two …...two aromatc rings for 7 index of hydrogen deficiency; one...

SPECIAL ONE DIMENSIONAL, AND TWO DIMENSIONAL NMR

A method which simplifies NMR spectrum

- Determine the exact frequency of HA or HX- Irradiate the system at the resonance frequency of HAinduces transitions between the two spin states of HA- Signal for HA disappears- HX can not distinguish the two spin states of HAand ‘sees’ a time average orientation of HA and a singlet for HX appears instead of a doublet.

A

B

Useful to simplify complex spectra and identify coupling partners

Signal for HA disappears

Signal for HX disappears

CHO

CH3

dd, H2

d, H3qdd

dqd

dd

H3qd, H2

qddqd

Only the threeMethyls can beidentified

1

2

3

4,56,7

8,9 10

R4C > R3CH > R2CH2 > RCH3

g-steric effectis shielding

Broad Band 1H decoupled 13C NMR Spectrum

Entire 1H range is irradiated as the 13C is observed

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Off Resonance Decoupled 13C Spectrum

Irradiation if 1H out side but near its resonance frequency (1000-3000 Hz)2J and above coupling removed1J coupling constant reducedPeak height increased

DEPT/INEPT SPECTRA

However, using DEPT/INEPT spectra we can ascertain how many protons are attached to each carbon.

The standard 13C NMR spectra are proton broadband decoupled.Each non-equivalent carbon appears as a single peak

DEPT (Distortionless Enhancement by Polarization Transfer)

INEPT (Insensitive Nuclear Enhancement by Polarization Transfer)

Multiplicity can be obtained from: 1) 1H Coupled 13C NMR Spectrum2) Off Resonance Decoupled 13C Spectrum3) DEPT/INEPT Spectrum

Run first the normal Broad band 1H decoupled13C NMR spectrum

1

2

3

4,56,7

8,9 10

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INEPT SPECTRUM OF CAMPHOR

Quaternary signals are not observed5

13C NMR SPECTRUM OF IPISENOL (75 MHz, CDCl3)

9

10

sp2 hybridized carbon atoms

sp3 hybridized carbon atoms

CDCl3

DEPT SPECTRUM OF IPSENOL

13C NMRALL SHOWN

↑ CH,CH3

↓ CH2

↑ CH

6

CDCl38,93,5

74 2 1,10

10

9

C

TWO DIMENTIONAL NMRThis is actually a misnomer because the plot is3-dimensional with the tops of the signals cut off to yield a 2-D contour plot.

HOMONUCLEAR CORRELATION SPCECTROSCOPY (H,H-COSY)

1D 1H NMR spectra are shown on both axesHelps to identify coupling partners2D contours are shown as diagonal peaks on a diagonal line at the intersection points when lines are drawn from peaks in the 1D spectra.Where coupling occurs among nuclei,contours (cross peaks, correlation peaks) occur as pairs outside the diagonal line.

1H NMR spectrum of ethyl acetate

2H3H3H

CH3 O

O

CH3

HH

Diagonal line

Diagonal peak

Cross peak

Cross peak(correlation peak)

CH3 O

O

CH3

HH

12

3

4.654.28 4.00

3.96 3.65 3.42

s

C13H12O310

.23

1H NMR spectrum (CDCl3, 300 MHz)

2.75

2.

64

17.3

3

CHCl3

7.49

07.

463

7.43

7

6.84

86.

845

7.05

77.

054

6.97

46.

971

7.08

47.

081

6.87

76.

819



2.74

6

2.64

12.

642

13C NMR spectrum (CDCl3, 75 MHz)20

4.91

8

32.1

75

25.2

29

168.

568

158.

319

13C NMR spectrum (CDCl3, 75 MHz)13

8.33

6

133.

259

132.

735 12

1.83

9

117.

452

113.

605

112.

962

111.

139

COSY spectrum

C13H12O3

[(2X13)+2]-12

2= 16

2= 8

Two aromatc rings for 7 index of hydrogen deficiency;one double bond for 1 index of hydrogen deficiency

Accounts for 10 C atoms

C=O (d 204.9) from 13C NMR2XAr-O- (d 168.6 & 158.3) from 13C NMR

2XCH3: dC 32.2 & 25.2; dH 2.75 &2.64 from NMR

C13H12O3

OH or COH10.230


2XCH32.75 & 2.64

17.3

31

CHCl3

HH

HOH

A B

AM

X

AMX spin system

8.1 Hz 7.8 Hz

0.9 Hz

8.1 Hz0.9 Hz7.

490

7.46

37.

437

6.84

86.

845

7.05

77.

054

6.97

46.

971

7.08

47.

081

6.82

26.

819


7.490-7.463=0.027ppm=0.027X3008.1 Hz

7.084-7.057=0.027ppm=0.027X3008.1 Hz

7.463-7.069 =0.394x300=118.2/8.1=14.527.069 – 6.835 =0.234x300=70.2/0.9=78

OH

HH

H

HH

HOH

A B

7.46 (t, J = 8 Hz)

6.86 (dd, J = 8, 1 Hz)

7.07 (dd, J = 8, 1 Hz)

CH3

OCH3

H

X

X

XX

J = 0.6 HzB

C=O 2XAr-OH

CH3

O

Ar-CH3

4X Ar-H

HH

HOH

HOH

CH3

COCH3A B

4X Ar --C

HH

HOH

HOH

CH3

COCH3A B

OH OHCH3

H

CH3 O

OH OH

HCH3

CH3

O

OH OH

CH3

O

CH3

C=O at d 204.9 ppm

No conjugation with aromatic ring

OH OH

CH3

CH3 O

1H NMR signals at d10.23 (s)17.33 (s)

OH OH

CH3

O

HCH3

HH

H

coupling

C=O at d 204.9 ppm:pushed out of plane;no conjugation with the ring

H,H-COSY Spectrum

OH OH

CH3

O

HCH3

OH OH

CH3

O

HCH3

HH

H

10.23 (s)17.33 (s)

2.75 (s)

2.64 (d, J =0.6 Hz )

OH OH

CH3

O

HCH3

HH

H

10.23 (s)17.33 (s)

2.75 (s)

2.64 (d, J =0.6 Hz )

6.97 (br d or m)7.07 (dd, J = 8.1, 0.9 Hz )

6.83 (dd, J = 8.1, 0.9 Hz )

7.46 (t, J = 8.1 Hz )

OH OH

CH3

O

HCH3

HH

H 1

2

45

8

9

10 36

7 11

13

12

1H,13C-COSY (HETROCOSY, HETCOR)1D 1H NMR spectrum is shown on one axis.1D 13C NMR spectrum is shown on the other axis.1J coupling correlation peaks (cross peaks) are shownat the intersection of 1H and 13C peaks.Helps to assign 1H data if 13C value is known. or to assign 13C data if 1H value is known.NO diagonal peaks are shown here.The modern method for observing correlation between1H and 13C peaks are called HMQC and HSQC(Heteronuclear Multiple Quantum Coherence).The sensitivity of 13C nuclei is increase 64 times.HSQC (Heteronuclear Single Quantum Coherence)HSQC better resolution along 13C axis, but the experiment involves more complex pulse sequence.

13

12

CD(H)Cl3

7

45

6

Quaternary carbon atoms can not be assigned from HMQC (or HSQC) spectrum

13127456

CHCl3

OH OH

CH3

O

HCH3

HH

H ?

?

121.8117.5

?

?

? ?

132.7

111.1 ?

25.2

32.1

13C ASSIGNMENT FROM HMQC SPECTRUM

LONG RANGE H,C-COSY SPECTRUMYields information on all kinds of carbon atoms,including quaternary carbons.

The term long range in H-C coupling applies for 2JCH and above.1JCH =125-250 Hz,2JCH is between -6 to 65 Hz

In the long range H,C-COSY, the experiment is optimized to observe only long range coupling.mainly 2JCH and 3JCHDirect C-H coupling may not entirely be eliminated.

NO diagonal peaks

HMBC (Heteronuclear Multiple Bond Connectivity).

11

134

1

OH OH

CH3

CH3 O

HX813

12

X X

12

HMBC (Heteronuclear Multiple Bond Connectivity)8-OH

8

103

6 547

6

45

7 2,9

Optimized for J=10 Hz

12 13

OH OH

CH3

O

HCH3

HH

H

HMBC CORRELATIONSOH OH

CH3

CH3 O

OH OH

CH3

O

HCH3

HH

H 168.6

113.6

121.8117.5

158.3

113.0

138.3 133.2

132.7

111.1 204.9

25.2

32.1

17.33 (s)

C=O at d 204.9 ppm

10.23 (s) Not observed

SCoAO

O

O O O O

O

OH OH

CH3

O

CH3

It is a natural product and the structure should be biogenetically explained

THE NUCLEAR OVERHAUSER EFFECT (NOE)

Spin-spin coupling takes place through bonds.The information is relayed by bonding electrons,leading to signal splitting.Magnetic nuclei can also interact through space,but the interaction does not lead to signal splitting.For protons close in space (2-4Å),when one is irradiated at its resonance frequencythe signal intensity of the other will either,increase (most of the time) or decrease.This is called the Nuclear Overhauser Effect (NOE)NOE falls rapidly as the inverse sixth power of distance between two nuclei.

1H COUPLED 13C NMR SPECTRUM

DOUBLE RESONANCE 13C NMR SPECTRUM

AN INCREASE IN INTENSITY IS DUE TO:i) Collapse of multiplets into singletsii) Nuclear Overhauser Effect (NOE)

Higher noise level

Lower noise level

Irradiation of protons increases the intensities of carbons in the neighborhood

APPLICATION

1H NMR spectrum of dimethylformamideThe methyl protons appear as two singlets at 20ºC

H N

O

CH3

CH3H N

+

O

CH3

CH3

A B

As the temperature gradually increased the two singlets coalesce into one singlet

H N

O

CH3

CH3

H-C=O

N+

CH3

CH3

How can we assign these two signals at room temp.?

H N+

O

CH3

CH3

B

Cis-CH3

trans-CH3

H-C=O

N+

CH3

CH3

a a

a b b a

b b1

4

2

3

H1

H2

W0W2Irr.

Irradiation of H1 (2→1,4→3)Population at levels 2 and 4 will be reducedPopulation at levels 1 and 3 will be increasedEquilibrium state is disturbed and relaxation (W) will take placePath W2 increases the population of 4 and decrease 1Population in 4 higher than 2 and 3 higher than 1Signal intensity of H2 will increase (Positive NOE)

H1 H2 -D

-D

+D

+D

SOURCE OF NOE

Path W0 increases the population of 2 and decrease 3population in 4 lower than 2 and 3 lower than 1Signal intensity of H2 will decreased (negative NOE)

Path W0 increases the population of 2 and decrease 3population in 4 lower than 2 and 3 lower than 1Signal intensity of H2 will decreased (negative NOE)

Relaxation by W2 is preferred by molecules with molecular weight between 100-400Relaxation by W0 is preferred by large molecules with molecular weight above 600Molecules with molecular weight between 400-600 may not show NOE, because both pathways take place, cancelling each other

NOE enhancement can reach up to 200%in 1H decoupled (broad band) 13C NMR spectra.In 1H NMR the maximum enhancement is 50%But usually between 1-20%.NOE is weak in CH3 group because each H can be relaxed by the other two nuclei.NOE is most easily detected by methine groups.

NOEs can be measured by integrating signals with the irradiation on and off, and measuring the difference in integration values.This can be measured if the enhancement is above 10%.

NOE DIFFERENCE SPECTRANOEs are more easily detected by subtracting in a computerThe normal spectrum from a spectrum taken with the irradiation signal on.All the unaffected signals simply disappear.The enhancement will be shown, and alsoan intense negative signal at the irradiating frequency.

H1 H2 H1 H2 H1 H2

=minus

NOE NOE differenceNormal spectrumH3 H4H4H3

Me

Me Me O

H

H

Me

H H

H

H

HH

1

2

34

5

67

8

910

Irr.NOE 5.5% 12.9%

irr2.9%

7.2%

5.0%

9.8%irr

8.7%

5.2%

S-cis rotamer

Me

Me Me CH3

H

H

O

H H

H

H

HH

More prominent rotamer

S-trans rotamer

Me

Me Me O

H

H

Me

H H

H

H

HH

S-cis rotamer

NUCLEAR OVERHAUSER AND EXCHANGE SPECTROSCOPY (NOESY)

NOE in two dimension. In NOESY The NOE between all protons can be observed simultaneously.NOESY resembles COSY, 1D 1H NMR spectrum is shown on one axis.1D 1H NMR spectrum is shown on the other axis.Diagonal peaks occur.Spatially close protons can be seen as a cross peaks.

abc

Me

Me Me O

H

H

Me

H H

H

H

HH 1

2

34

5

6

78 9 10

3, 2

From NOESY Spectrum

abc

Me

Me Me O

H

H

Me

H H

H

H

HH 1

2

34

5

6

78 9 10

3

4

1

2

3

4

1

2

3

4

1

2

+ve J -ve J -ve J

3+41+212 214 3 43

+VE AND –VE J VALUES

+ve J

1H 13C 13C 13C

AA

A

BB

BCC

C

DD

D

special one dimensional, and two …...two aromatc rings for 7 index of hydrogen deficiency; one...

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