Report

TWO-Dimensional NMR spectroscopy

(Practical Applications and Spectral Analysis) A report submitted in (partial) fulfilment of the requirements for the completion ofChem993:

Awad Nasser Albalwi

Supervised by

Dr.Michael Kelso & Dr.Wilford Lie

School of Chemistry

University of Wollongong

2

Table of Contents

I COVERING INFORMATION…………………..1

II CONTENTS…………………………………..……………2

1 INTRODUCTION…….………………..…………….. 3

1.1 APT………………………………………………………………...5

1.2 DEPT …………………………………..………………………. 5

1.3 COSY …………….………………………….….………….….6

1.4 NOESY.….….….………………………………..………….….6

1.5 TOCSY ……………………………………………………...…7

1.6 HMBC …………………………………………………….……8

1.7 HSQC ……………………………………...............……..9

1.8 the project Aims ……………….………………………10

2 The procedure………………………………………..…11

3 Results…………………………..…………………….….…12-18

4 Discussion……………………………………..……….…19-22

5 Conclusion …………………………………………………..22

6 Acknowledgments ………………………..……………22

7 References ……………………………………………………22

8 Index

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1. Introduction

Over the past fifty years nuclear magnetic resonance (NMR), has become the

preeminent technique for determining the structure of organic compounds. It is the

only spectroscopic method with a complete analysis and interpretation of the entire

spectrum is normally expected., nmr is non-destructive, and with modern instruments

good data may be obtained from samples weighing less than a milligram.

All nuclei that contain odd number of protons or neutrons poeeess an observable

nuclear magnetic dipole, magnetic moment (µ) and angular momentum (J). In the

presence of external static magnetic field (B) , the angular frequency (w) is linearly

dependent on (B) 12

W=

The nuclei of many elemental isotopes have a characteristic spin. Isotopes that are

useful to organic chemists are 1H, 13C, 19F and 31P, all of which have nuclear spin quantum

number (I) I = 1/2. The characteristic spin can be thought of as a small magnetic field,

and will cause the nucleus to produce an NMR signal in a static magnetic field. The

intensity of a peak observed in an NMR spectrum is related to the transition

probability between the energy level -1/2 and +1/2.

Chemical Shift

Different frequencies of peaks are observed in NMR spectra obtained from a sample

containing many nuclei in each molecule. Unlike infrared and uv-visible spectroscopy,

where absorption peaks are uniquely located by a frequency or wavelength, the location

of different NMR resonance signals is dependent on both the external magnetic field

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strength and the frequency. Since no two magnets will have exactly the same field,

resonance frequencies will vary accordingly and an alternative method for

characterizing and specifying the location of NMR signals is needed. The resulting shift

spectrum is compared with those of model compound. As the molecular complexity

increased, the 1D NMR can be improved by running the experiment at higher and higher

magnetic field strength. It thus allows interpretation of complex first order –like

spectra. Spectra obtained at highest available magnetic fields are obscured and signal

are overlapped for complex molecules

2D NMR Spectroscopy

2D NMR offer a new way to resolve even higher overlapping spectra into interpretable

multiplet and permit a chemical shift assignment to be made in a simple and direct

manner by speading resonances out into two dimensions and thereby increases the

resolutions.

There are two broad types of experiments that are commonly used in 2D NMR.

1 Correlation experiment (COSY)

2 Nuclear overhauser effect spectroscopy (NOESY)

Others are attached proton test (APT), (DEPT), (TOCSY), (HMBC), (HSQC)

In 2D NMR one dimension gives us J coupling information while the other dimension

gives chemical shift information. This type of experiment is called homonuclear J-

Resolved 2-D NMR. There is also heteronuclear J-resolved 2-D NMR which uses a spin

echo sequence and techniques similar to those described above. The tool is useful in

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well resolved spectra. 2-D measurement can be performed simultaneously for all line in

a spectrum.

1-1-(APT) The Attached Proton Test (APT) experiment is a common way to assign C-H multiplicities

in 13C NMR spectra. It provides the information on all sorts of carbons within one

experiment. Depending on the number of hydrogens bound to a carbon atom, n, CHn spin

vectors evolve differently after the initial pulse. If the delay is set to 1/J, CH and CH3

vectors have opposite phases compared to C and CH2. Therefore, the phase of CH and CH3

peaks is 180° different from C and CH2 peaks. This example demonstrates the basic

procedure of double resonance 1D NMR data acquisition and processing on spectrometers.

1-2-The DEPT: Distortionless Enhancement by Polarization Transfer

The DEPT the method has become one of the most important techniques available to the

NMR spectroscopy for determining the number of hydrogens attached to a carbon atom.

The pulse sequences involves complex program of pulses and delay times in both the 1H &

13C . The result of pulses sequences for DEPT is that carbon atoms with one ,two and three

attached hydrogens exhibit different phases as they are recorded. The phases of these

carbon signals will depend on the duration of the delays that are programmed into the

pulse sequence. In DEPT spectrum , the quaternary carbons , which have no attached

hydrogens , give no signal .

Fig.1 :The pulse sequences.2.

6

1-3-COSY Correlation Spectroscopy

Cosy is one of the first 2D NMR spectra reported and It is useful for studying relatively

small molecules. COSY arise from coupled spins that in the corresponds to protons

separated by two or three bonds .The pulse sequence for the cosy is two 90◦ pulses

separated by an evolution time followed by the acquisition of the NMR signal3.

Fig.2:The pulse sequence for the cosy2.

2D COSY technique allows to walk-through the spectrum for the assignment of

neighbouring protons.3

1-4-NOESY: NUCLEAR OVERHAUSER ENHANCEMENT SPECTROSCOPY

The NOESY spectrum uses the nuclear Overhauser effect (nOe) to provide information

about which proton (typically) resonances are from protons which are close together in

space. This is distinct from COSY-type spectra which use the J-coupling interaction to

report on which proton resonances are located on the same or adjacent carbon nuclei.

While it is true that most protons which are on adjacent carbons are close enough to give

a NOESY cross-peak, it is not necessarily true that protons which give a NOESY cross-

peak are J-coupled3.

In NOESY , all interproton nOe effects appear simultaneously, and the spectral overlap is

minimized due to the spread of the spectrum in tow dimensions. The pulse sequence for

NOESY is shown in fig. Figure has shown that

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1-5-TOCSY:

The TOCSY allows for identification of all the protons through bond correlations by

carbons and nitrogens1. The pulse sequence for the TOCSY contains a train of pulses in

mixing period. The most popular mixing for TOCSY is MLEV-17 which consists od a train of

[(90◦)x-(180◦)x-(90◦)x] pulses to lock nuclear spins onto the x and y axis 2.

Fig.4: The pulse sequence for the TOCSY.1

Ramadan,S & Mountford,C 2009,Annual reports on NMR spectroscopy,vol.65, Elsevier

Ltd.

Fig.3: The pulse sequence for NOESY.1

8

1-6-HMBC :Heteronuclear Multiple-Bond Connectivity spectra:

The HMBC spectra provides information about long-rang 1H coupling interactions with 13C

nuclei. The HMBC likes the HMQC is also an inverse for example, 13C population

differences are measured indirectly through their effects on 1H nuclei. This leads to make

HMBC more sensitive. The pulse sequences used 1JCH correlations is shown in( fig.5)1.

The first 90° 13C pulse serves to remove the one-bond 1JCH correlation so that cross peak

due to direct connectivities do not appear, allowing long-rang 1H-13C connectivities to be

recorded. The second 90° 13C pulse creates zero and double quantum coherences , which

are interchanged by the 180° 1H pulse. After that the last 90° 13C pulse, 1H signals

Fig.5:The pulse sequence for the HMBC

experiment.

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resulting from 1H-13C multiple-quantum coherences are modulated by 13C chemical shifts

and homonuclear proton couplings. The HMBC allows to assign protonated nuclei such as,

quaternary carbons after identifying their neighbouring protons1.In addition The HMBC

spectrum helps in confirming the assignments related to protonated nuclei2

Fig5a: long-rang 1H-13C connectivities

1-7-HSQC: The Heteronuclear Single Quantum Coherence:

The Heteronuclear Single Quantum Coherence (HSQC) experiment is used frequently in

NMR spectroscopy of organic molecules and is of particular significance in the field of

protein NMR . The resulting spectrum is two-dimensional with one axis for 1H and the

other for a heteronucleus (an atomic nucleus other than a proton), most often 13C or 15N.

The spectrum contains a peak for each unique proton attached to the heteronucleus being

considered. Thus, if the chemical shift of a specific proton is known, the chemical shift of

the coupled heteronucleus can be determined , and vice versa. The HSQC allows to

identify CH2 pairs that are attached to the same carbon2 (fig.6 ).H-C HSQC spectra are

usually optimised observe only peaks that are correlated to spin-spin coupling around 140

Hz2.

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Fig.6:CH2 pairs that are attached to the same carbon2.

HMQC Spectra

1-8-Aims

The aims of this project are to:

Provide essential knowledge and understanding of two-dimensional (2D) NMR

spectroscopy

Using 2D NMR knowledge to study selected areas in chemistry, biology and medical

research usually related to the investigation, confirmation and determination of

molecular contents, structures, and interactions.

Improve the practical laboratory knowledge and experience of master students

using high-resolution NMR experiment and NMR data processing.

Fig.7:The pulse sequence employed for the HMQC.1

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2-The procedure:

The sample No.24709 was obtained from the administrator. The sample was then

dissolved in a little amount of CDCl3 . The mixture was transferred into the NMR thin

glass tube. The NMR tube was then inserted into the 500MHz NMR module.

The running NMR was done using the given NMR-training-venus1005 manual.

The processing the data was done using the NMR program . The 1D proton, APT & DEPT of

1D Carbon, COSY ,NOESY ,TOCSY ,HMBC and HSQC were analysed to distinguished the

unknown sample from the other two sample and then each Carbon and proton were

identified clearly using the obtained spectrums.

Sample.1

Sample.2

Sample.3

Fig.9.the structure of three possibilities of unknown compounds.

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3-The results:

Table.1 Proton Spectrum Assignments.

No Ppm split Integration Assign’t Notes

1 4.18 S 1 H2 Alphatic hydrogen group of CH

2 4.06 S 3 H18 Aliphatic hydrogen group of OCH3

3 3.39 S 1 H9a Aliphatic hydrogen group of CH

4 3.04 M 1 H5.1 Aliphatic hydrogen group of CH2

5 3 M n/a H10 Aliphatic hydrogen group of )CH

6 2.93 M 1 H5.2 Aliphatic hydrogen group of CH2

7 2.62 D 1 H7 Aliphatic hydrogen group of CH

8 1.99 S 3 3*H16 Aliphatic protons , CH3

9 1.88 D 1 H1a

Aliphatic hydrogen group of CH2

10 1.84 M 1 H6a Aliphatic hydrogen group of CH2

11 1.74 M 1 H6b

12 1.73 M 1 H9 Aliphatic hydrogen group of CH

13 1.64 M 1 H1b Aliphatic hydrogen group of CH2

14 1.50 M 2 2H2` Aliphatic hydrogen group of CH2

15 1.35 M 1 H1` Aliphatic hydrogen group of CH2

16 1.30 d 3 3*H17 Aliphatic proton,CH3

17 1.262 d 2 H3` Aliphatic hydrogen group of CH2

18 1.186 2 H1` Aliphatic hydrogen group of CH2

19 0.84 S 3 3*H4` Aliphatic protons,CH3

13

Table.2:Carbon Spectrum Assgnments. Peak No Ppm Group Assign’t

1 169.68 Q – C15 -(C=O)

2 162.80 Q C13

3 148.41 Q C11

4 127.82 Q C12

5 112.67 Q C8

6 98.51 Q C14

7 82.80 Q C3

8 78.51 CH C2 connected to electronegative atom-O

9 77.375 solvent

10 60.89 CH C9a

11 58.80 CH C18 (OCH3)

12 49.89 CH C9

13 47.55 CH C7

14 47.51 CH2 C5 connected to electronegative atom N

15 34.52 CH3 C10

16 33.23 CH2 C1`

17 31.57 CH2 C1

18 27.27 CH2 C2`

19 26.62 CH2 C6

20 23.09 CH2 C3`

21 18.30 CH3 C17

22 13.95 CH3 C4`

23 9.12 CH3 C16

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Table.3:HSQC Proton spectrum Assignment

HSQC Peaks

N0

Assign’t Notes

1 H18 –C18 Assigned after the proton was identified

2 H2-C2 Assigned after the proton was identified

3 H16-C16 Assigned after the proton was identified

4 H17- C17 Assigned after the proton was identified

5 H10-C10 Assigned after the proton was identified

6 H14`-C4` Assigned after the proton was identified

7 H3`-C3` Assigned after the proton was identified

8 H1`-C1` Assigned after the proton was identified

9 H2`-C2` Assigned after the proton was identified

10 H5-C5 Assigned after the proton was identified

11 H9a-C9a Assigned after the proton was identified

12 H9-C9 Assigned after the proton was identified

13 H1-C1 Assigned after the proton was identified

14 H7-C7 Assigned after the proton was identified

15 H6-C6 Assigned after the proton was identified

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Fig.10:APT spectrum.

Fig.11: proton spectrum

3H18

H2

H9

3H4`

3H17

1H7 2H5

3H16

2H1

2H6

2H1` 2H3`

2H2`

C5

C18

C13

C15

C11

C12

C8 C14

C3

C9

C7 C10

C9a

C2

C1

C1`

C3`

C2`

C6

C17

C4`

C16

H9a

H10

16

Fig.12:proton spectrum.

Fig.13: HSQC spectrum.

H2

H18

H9a

H5

(C2-H2) C18-H18

(C9a-H9a

(C5-H5) (C10-H10)

(C7-H7)

(C9-H9)

(C1-H1)

(C1`-H1`)

(C6-H6)

(C2`-H2`)

C3`-H3`

C17-H17

C4`-H4`

C16-H16

H1

H6

H9

H2`

H1`

H17

H3` H16

H7

H10

H5

H18

H9a H2

C9a

C18

H7

17

Fig.14:COSY spectrum.

Fig.14:TOCSY spectrum

H4`-H3`

H4`

H3`

H10-H17

H2-H1

H1

H9a

H2-H9a H6

H9a-H9

H7-H6

H7

H18

H2

H10

H5

H7

H4`

H7

H18

H9a

H7

H5

H10

H16

H17

C4

`

18

Fig15. NOESY spectrum.

H2

H18 H9a

H7

H10

H5

H16

H4`

H17

H2`

H1

H1`

H3`

H6

H9

H5-H9a

H18-H16

H9-H9a

H1-H9a

H17-H9a

H1`-H2

H2`-H2

H1-H2

H16-H18

H17-H18

19

4-Discussion:

The APT spectrum shows that the unknown compound has twenty two Carbons involving

seven quaternary carbons , five Carbons of CH group , six Carbons of CH2 groups as well

as four Carbons of CH3 group. The APT has distinguished the unknown compound from

Other compounds and clearly has shown the structure is exactly same for sample

No.24709 .

Figure.16 HMBC , COSY and TOCSY system.

Initially, From APT experiment, C15 can be identify at the highest chemical shift (169

ppm) ,because it is the group of Carbonyl . The C18 can be identified at 58.8ppm by APT

and proton integration (3H) ,because this Carbon is for group of (OCH3) which is likely to

be shifted toward down field than other three groups of CH3 as a result of binding to

electronegative atom (Oxygen) (fig.10&11) . The protons 3H 18 of group (OCH3) was then

identified by using HSQC spectrum at 4.06ppm. COSY experiment has shown these

protons(3H18) are not coupling with other neighbouring protons which confirm these

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protons belong to (OCH3). Furthermore, HMBC has indicated clearly, that there is only

correlation relationship between 3H18 and C13.

After proton H18 was identified , the carbon No.13 was identified at 162.80ppm using

HMPC experiment, because there was only one correlation relationship between (H18-

C13).In addition, The C13 is likely to be shifted toward down field than C14 because of an

effect of electronegative atom (Oxygen) attached to it .

After C13 was identified, The protons N0.16 were identified at 1.99ppm as a signal peak

with three integration .these protons (3H16) have shifted toward downfield as a result of

CH3 group being connected to sp2 Carbon (C14).In addition, HMBC experiment has

confirmed the correlation relationship between H16 & C15. Moreover, COSY experiment

has indicated that these protons (3H16) has no coupling with neighbouring protons which

distinguish these protons of CH3 from other two remaining (17 &4`) CH3 groups. The

TOCSY system has shown there are only two spin coupling between (H16-H18) and (H16-

H17). The C16 was then identified at 9.12ppm by HSQC spectrum.

The C14 was identified at 98.51ppm using HSQC, because HMPC has shown only one

strong correlation relationship between H16&C14.

The quaternary carbons C11 & C12 were identified at 148.41&127.82ppm respectively in

relation to H16 . The HMPC experiment has shown correlation relationship between (H16-

C12) which is stronger than (H16-C11). On the other hand, HMBC has shown the correlation

between (H10-C11) is stronger than (H10-C12) . From above statement, it can be said that

the closer Carbon to C10 is C11. In addition, the HMBC has shown clearly the correlation

between (H17-C11) which did not exist in case of C12.

Using HMPC ,The 3H17 of CH3 group was identified at 1.30ppm from the correlation

relationship between (C11&H17) as well as proton integration which confirmed the

presence of three protons. The C17 was then identified by using HSQC experiment. After

C17 was identified , The H10 was suggested to be at 3ppm because of HMBC confirmed

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one relationship between one proton ( H10) & C17 as well as COSY confirmed the (H10-

H17) correlation relationship. The HMBC confirmed that H10 has correlation relationship

with C11 and C12. The C10 was then identified by HSQC at 34.52ppm.

COSY has shown correlation between H10 and H9 (at 1.73ppm). HMBC has indicated that

H9 is correlated with quaternary carbon C8 and C17. The C9 was then identified at

47.55ppm using HSQC experiment. The H9a was then identified at 2.62ppm because

COSY experiment has shown the correlation between H9a and H9. Moreover, the COSY

has shown clearly strong correlation relationship between H9a & H1 as well as weak

correlation between H9a and H2. The C9a was then identified at 49.89ppm. from COSY

also H1a and H1b were identified at 1.88&1.64ppm. H1 has correlation with H9a and H2.

The C1 was then identified at 31.57ppm using HSQC. The proton No.2 was located at

4.18ppm based on (H1-H2) and (H9a-H2) COSY correlation relationship. HMBC has

confirmed that there was correlation between C8&H2). The C2 was then identified at

78.51ppm.

The TOCSY experiment has confirmed the Chain of protons of H2,H9a,H10, H1a,H1b and

H9 (fig.16).

The remained quaternary carbons C8 & C3 are present at 112.67 and 82.80ppm

respectively. The C8 is shifted toward down field than C3 as a result from the strong

effect of two electronegative atoms with Oxygen connected to it in contrast of an

effect of Nitrogen connected to C3. Furthermore, HMBC has confirmed later the

correlation relationship between (H9a-C8), (H2-C8) and (H7-C8) . In addition, HMBC has

confirmed later the correlation between (H2`-C3) and (H5-C3).

The last Carbon of group CH3 (C4`) is located at 13.95ppm and its protons is at 0.84 base

on HSQC experiment. The protons H4` is likely to see protons H3` which is confirmed by

COSY experiment at 1.26ppm. The C3` is located at 23.09ppm regarding to HSQC

experiment. After identification of C3` , the H2` was identified at 1.5ppm based on the

22

correlation relationship between C3` & H2`, HMBC & COSY spectrums. Then , the C2`

was identified at 23.09ppm. HMBC has shown the correlation between 2H2` and C3.

The H1` was identified at 33.23ppm with respect to COSY correlation between H1` and

H2`.

The TOCSY indicated that there was a system between H1`,H2`,H3` and H4` (fig.16)

The Carbon C5 of CH2 group is mostly to be at the largest chemical shift (47.51ppm)

among other CH2 groups as a result of an effect of Nitrogen atom. And the two protons

of C5 were located at 3.04&2.93ppm using HSQC. This was confirmed by the correlation

relationship between H5 & C3 using HMBC. The 2H6 were located at 1.84&1.74ppm using

COSY correlation between H5 and H6 as well as H6 and H7. The HMBC has also shown

correlation between H6 and quaternary C8.The C6 was then identified at 26.62ppm. The

last C of group CH was presence at 60.89ppm and its proton at 2.62ppm. The HMBC has

shown strong correlation between H7 and C8 as well as C3.

The NOESY has shown that there is a system of 2H5, 2H6 and 2H7 . (fig.16)

5-Conclusion:

In this work, the unknown compound was successfully distinguished from other two

compounds and then each carbon and protons were identified using 1D & 2D of NMR

techniques . APT,DEPT, COSY,NOESY TOCSY, HMBC and HSQC experiments were used

in this project. In addition to this it was also found that each spectrum was supported by

another spectrum in identifying every single atom. This gave high confidence with respect

to analysing the results.

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6-Acknowledgments

first of all I would like to thank Allah for his blessing and guidance without which I

couldn’t finish this report .I would also like to thank the University of Wollongong ,School of

Chemistry for computational facilities , Academic resources and on NMR facilities. I

would also like thank Dr Wilford Lie for helpful discussions ,training on NMR techniques

and continuous guidance, support and motivation .I would also like to thank Dr Michael

Kelso, subject coordinator (CHEM993) for his guidance and support which was useful for

doing this report. Lastly but in no way the least I would like to thank my family and

friends for their continuous support, prayers and love.

7. References

1 Rahman ,A & Choudhary,M , 1996, Solving problems with NMR spectroscopy, Academic Press,INC , San Digo.

2Wilford Lie, 2010, Chem993 NMR Spectroscopy, lecture notes, UOW 3 Ramadan,S & Mountford,C 2009,Annual reports on NMR spectroscopy,vol.65, Elsevier Ltd. 4 William R. Croasmun and Robert M.K. Carlson,

Two-dimensional NMR spectroscopy : applications for chemists and biochemists

5 http://www.chem.uic.edu/nmr/downloads/Avance-NOESY-Guide.0702.pdf

24

The INDEXT