1st Semester 2017 and 2nd Semester 2018

Junior Sophister

Organic and Inorganic Practicals

Analytical Spectroscopy Course - NMR : Prof. Thorri Gunnlaugsson

NMR Facility : Dr John O’Brien, Dr Manuel Ruether

NMR analysis in Cocker lab : Lab Demonstrators

Revision from Senior Freshman Year

Organic Spectroscopy course : Prof. Isabel Rozzas

- NMR dealt with in lectures 2 to 5

- Topics included :

Hydrogen (Proton) NMR

Carbon 13 NMR

MRI - imaging using NMR

• NMR is the major analytical technique used to identify and characterise

materials today.

• NMR is used for :

– Identification of nuclides and their environment

– Conformational analysis

– Interactions of molecules

– Monitoring processes and reactions

• The main experimental methods are :

- FT NMR (superconducting magnets)

- Dynamic NMR, MRS, MRI

Introduction

Solution NMR

• Most common method by which NMR is carried out

• Materials and substances (liquids or solids) are dissolved in appropriate solvents

– deuterium replaces hydrogen in the solvent

• Many and varied experimental techniques in NMR, which are available to the user

• Usefulness is the fact that the nuclear spins under the influence of the magnetic field are

dependent on the molecular environment

• Position of peaks (resonances) are readily identifiable and information from peak

positions are reproducible from molecule to molecule

NMR of Liquids

• Applications :

– Academic (research, teaching)

– Industrial (chemical, pharmaceutical, medical, food, agriculture, ……)

In the fields of :

– Chemistry, Biochemistry, Biology, Physics, Pharmaceutical Chemistry, Medicine,

Genetics, Geology, Engineering, Forensic Science etc.

NMR Spectrometers

• Current equipment in TCD :

- 800 MHz (18.8 T) instrument for Biochemistry i.e. large protein work

- 600MHz (14.1T) solution instrument - Chemistry

- 400MHz (9.4T) solution instruments - Chemistry (2), TBSI (1)

• Major Nuclides

- 1H, 13C, 31P, 19F, 15N

- 2D, 11B, 17O, 29Si, 14N, 117Sn, ….

The 800MHz NMR

The Chemistry NMRs

600MHz NMR

400MHz NMRs

The biggest available NMR - 1000MHz

Yours for Millions of Euro !

Very sensitive

This NMR would need its own

warehouse in the middle of the

Cricket pitch because of the big

magnetic stray field

as of 2016 there is a new

generation of shielded magnets

at 1000Mhz

coming - 1200MHz NMR

A real NMR study

• Thyrotropin Releasing Hormone (TRH)

• TRH is a neuropeptide which is involved in some CNS disorders

• TRH has a function in the repair of brain and spinal disorders

Using NMR to identify a Hormone

A Case Study in NMR Methods

The TRH molecule is small peptide

• building block of a peptide is an amide linkage

C

O

N

H

C

H

C

CH2

O

N

H

TRH

• a Tripeptide with an amino terminated proline

• p-Glutamic acid Histidine Proline

N

C NH2

O

HN CH C

CH2

O

N

NH

C

O

NH

O

NMR of TRH

• The aim is to identify and assign all the positions of the atoms in the peptide

• Verify that the putative structure is correct

The tools

• Proton (Hydrogen) NMR

• Carbon-13 NMR

• Nitrogen-15 NMR

Proton (Hydrogen) NMR

• Position of the signal – resonance

• Hydrogen - hydrogen interaction

- spin coupling

• Ratio of the amount of proton signals

- integration

Carbon-13 NMR

• Carbon-13 is the active NMR nuclide

- only 1% of all Carbon

• Position of the Carbon signals

• Other Carbon-13 experiments to determine the

type and number of hydrogens attached or none

e.g. C, CH, CH2, CH3

Proton (HYDROGEN) NMR

( p p m )

1 . 02 . 03 . 04 . 05 . 06 . 07 . 08 . 09 . 01 0 . 01 1 . 01 2 . 0

Solvent

DMSO-d6

Water

NH or OH

Aromatic H , NH, OH Aliphatic CH, CH2, CH3s

CH2

CH2CH

N

H2C

C NH2

O

HN CH C

CH2

O

N

HC NH

CH

C

O

CH

H2C

H2C

CNH

O

1H NMR with peak labels and integration

(ratio of signal volume with respect to one another) 0

.9

76

6

1.

61

77

1.

00

00

1.

18

24

1.

55

20

0.

99

85

0.

81

73

1.

03

66

0.

82

07

6.

78

09

1.

93

72

8.

03

64

In

te

gr

al

11

.9

14

3

8.

18

14

7.

79

81

7.

53

09

7.

18

90

6.

97

01

6.

90

17

4.

63

54

4.

20

44

4.

06

20

3.

56

52

3.

32

37

2.

95

24

2.

81

06

2.

51

20

( p p m )

1 . 02 . 03 . 04 . 05 . 06 . 07 . 08 . 09 . 01 0 . 01 1 . 01 2 . 0

1.

61

77

1.

00

00

1.

18

24

1.

55

20

8.

18

14

7.

79

81

7.

53

09

7.

18

90

6.

97

01

6.

90

17

( p p m )

7 . 07 . 58 . 0

0.

99

85

0.

81

73

1.

03

66

4.

63

54

4.

20

44

4.

06

20

( p p m )

4 . 04 . 4

Integrals (value and trace)

Peak labels

solvent

3 x CHs

NH histidine

2x NH, 1x NH2

2x CH histidine

Carbon-13 NMR

( p p m )

2 03 04 05 06 07 08 09 01 0 01 1 01 2 01 3 01 4 01 5 01 6 01 7 01 8 0

Carbonyl –C=O

Aromatic C

Aliphatic CH s CH2 s

SOLVENT N

C NH2

O

HN CH C

CH2

O

N

NH

C

O

NH

O

More Types of Carbon-13 NMR

Carbon DEPT (distortionless enhancement by polarisation transfer)

3 04 05 06 07 08 09 01 0 01 1 01 2 01 3 01 4 01 5 01 6 01 7 0

3 04 05 06 07 08 09 01 0 01 1 01 2 01 3 01 4 01 5 01 6 01 7 0

( p p m )

CH2s

CHs

CHs

C -C=Os

Carbon DEPT 135°

13C Carbonyl (C=O) and methine (CH) signals

( p p m )

2 03 04 05 06 07 08 09 01 0 01 1 01 2 01 3 01 4 01 5 01 6 01 7 01 8 0

( p p m )

1 7 01 7 21 7 41 7 61 7 8

( p p m )

5 05 25 45 65 86 0

CH

N

C NH2

O

HN CH C

CH2

O

N

NH

C

O

CH

CNH

O

4 x C=O

CH

CH CH

Note the extra signals from an isomer *

and a rotormer

*

More NMR tools - 2D

• CH and NH correlation – through bonds

- direct (one) bond or further out

• HH correlation - through bonds, looks more complex

- connections are the off diagonal signals

• Through space connections for HH - NOe, NOESY

• Selective 1D versions of the 2D experiments

13C-1H Correlation (edited HSQC)

Links carbon signals to hydrogen(s)

CH2

CH2CH

N

H2C

C NH2

O

HN CH C

CH2

O

C

N

HC NH

CH

C

O

CH

H2C

H2C

CNH

O

13C axis

1H axis

15N-1H Correlation (15N HSQC)

NH

NH

NH

NH2

N

C NH2

O

HN CH C

CH2

O

N

NH

C

O

NH

O

15N axis

1H axis

NH2

NH NH

CH

CH

Combining NH and CH 2D Correlation Experiments

CH2

CH2CH

N

H2C

C NH2

O

HN CH C

CH2

O

C

N

HC NH

CH

C

O

CH

H2C

H2C

CNH

O

Peak overlap

Problems in 1H spectra

0.

99

85

0.

81

73

1.

03

66

0.

82

07

6.

78

09

1.

93

72

8.

03

64

In

te

gr

al

4.

63

54

4.

20

44

4.

06

20

3.

56

52

3.

32

37

2.

95

24

2.

81

06

2.

51

20

( p p m )

1 . 41 . 61 . 82 . 02 . 22 . 42 . 62 . 83 . 03 . 23 . 43 . 63 . 84 . 04 . 24 . 44 . 64 . 8

Peak overlap

4 x CH2s

Complex spin- spin coupling

CH2 CH2

3 X CHs

CH correlation in aliphatic region

3x CH

CH2

CH2

Two separate proton signals

for one carbon 46.7

3.56, 3.20

30.3

2.95, 2.80

Identifying the remaining methylene peaks

Possible to identify the proton

peaks directly for the 4

overlaping CH2s

13C axis

1H axis

2.01, 1.86 2.11, 2.06

1.83, 1.78

2.95, 2.80

2.22, 1.85

25.0

24.2

29.0

29.2

30.3

Gathering the Information in a Peak table with assignments for carbon and nitrogen attached to protons

d

13C

ppm

d 15N

ppm

d 1H

ppm

CH aromatic

134.9

-

6.97

CH

113.7

-

7.53

CH aliphatic

60.1

-

4.21

CH

55.2

-

4.05

CH aliphatic

51.2

4.63

CH2

46.7

-

3.56, 3.20

CH2

30.3

-

2.95, 2.80

CH2

29.2

-

2.01, 1.86

CH2

29.0

-

2.11, 2.06

CH2

25.0

-

2.22, 1.85

CH2

24.2

-

1.83, 1.78

NH

-

166.6

11.92

NH

-

122.2

8.21

NH

-

121.1

7.80

NH2

-

105.2

8.18, 6.90

HH correlation - links local protons

HNHC

CH2

NH

histidine aliphatic fragment

CH CH2

H2C

CNH

CH

H2C

C

O

NH

O

CH

CH2

N

NH

C

O

N

CHCH2

CH2

H2C

CH2

C NH2

O

HH Correlation - Links to local protons (diagonal is the 1D spectrum)

CH

HH COSY

Combined through bond (HH) and space (NOE) correlation

CH2

CH2CH

N

H2C

C NH2

O

HN CH C

CH2

O

C

N

HC NH

CH

C

O

CH

H2C

H2C

CNH

O

CHhis CH2 pglu

NHhis CHpglu

HH COSY and NOESY

Selective through bond and through space NMR

experiments

• These are 1D analogues of 2D NMR experiments

• They are very powerful tools for elucidation when appropriate

• There are two classes of experiments

– through bond - selective TOCSY or COSY

– through space - selective NOE or ROE

• Normally quicker to run than their 2D equivalent

• Extract specific and often detailed information

• Requirement that individual signals are accessible

Selective HH correlation experiments - through bond connections

same information as 2D TOCSY experiments but with better resolution

CH2

CH2CH

N

H2C

C NH2

O

HN CH C

CH2

O

C

N

HC NH

CH

C

O

CH

H2C

H2C

CNH

O

correlated spins from the separate CHs peaks

(found in 4-5ppm region)

Histidine -NH-CH-CH2-

Proline - no NH

-CH-CH2-CH2-CH2-

p-glu -NH-CH-CH2-CH2-

4.06 ppm

4.21 ppm

4.63 ppm

Selective Nuclear Overhauser ( Noe) experiments

- through space connections

*CH

*CH

*CH

* Irradiated CH peak

proline

p-glu

histidine

NH2 8.18

NH 8.19

CH 6.97

NHs 7.80, 8.19

CH2

CH2CH

N

H2C

C NH2

O

HN CH C

CH2

O

C

N

HC NH

CH

C

O

CH

H2C

H2C

CNH

O

Long range CH correlation (HMBC) Very useful - links proton to carbon with NO proton(s) attached

Carbonyl peaks

-C=O

13C axis

1H axis

177.4

173.7

171.9

170.0

p-glu

His CH - two –C=O links

NH CH

H2C

CNH

CH

H2C

C

O

NH

O

CH

CH2

N

NH

C

O

N

CHCH2

CH2

H2C

CH2

C NH2

O

Long range CH correlation (HMBC)

-C=O to CH links -NH2 to -C=O

p-glu

pro

his

CH

N

C NH2

O

HN CH C

CH2

O

N

HC NH

CH

C

O

CH

CNH

O

173.7 to 6.90 and 8.18 ppm 4.63 to 170.0 and 171.9 ppm

4.21 to 173.7 ppm 177.4 to 4.06 ppm

177.4

170.0

171.9

173.4

4.63 4.21 4.06

Proton NMR assignments

N

C

H2N

O

NH

CH

C

CH2

O

NNHHN

C

O

O

7.53

6.97

6.90,8.18

4.63

4.21

4.06

3.56, 3.20

2.95, 2.80

11.92

8.197.80

2.22, 1.85

2.11, 2.06

2.01, 1.86

1.83, 1.78

Carbon-13 , Nitrogen-15 NMR assignments

N

C

H2N

O

NH

CH

C

CH2

O

NNHHN

C

O

O

177.4

173.7

171.9

170.0

135.9134.9

113.7

30.351.2

55.2

60.1

29.2

29.0

25.0

24.2

46.7

105.2

121.1

122.2

166.6

15N assignments 13C assignments