Lab Report on the NMR Spectroscopy of Organic Molecule

The lab report below was submitted as part of the coursework for CM2102 Spectroscopic Applications. Please do not plagiarise from it as plagiarism might land you into trouble with your university. Do note that my report is well-circulated online and many of my juniors have received soft copies of it. Hence, please exercise prudence while referring to it and, if necessary, cite this webpage.

Aims

To obtain an NMR spectrum of a liquid mixture consisting of ethyl acetate, p-tolualdehyde and cyclohexene in CDCl₃ solvent, assign each peak of the spectrum to the different molecules, and to determine the concentration ratio of the organic compounds in the mixture.

List of reagents and instruments

Bruker 500 Ultrashield NMR spectrometer connected to computer

Sample of ethyl acetate, p-toulene and cyclohexene dissolved in CDCl₃ solvent

Experimental procedures

1.       Fit the NMR tube into the spinner

2.       Wipe the NMR tube

3.       Click “Lift-On/Off” button  to turn on gas flow

4.       Ensure that air is blowing out, load the NMR tube into the spectrometer

5.       Click “Lift-On/Off” button to turn off gas flow

6.       Type “dir”

7.       Select “Standard ¹H”

8.       Type “edc”

9.       Enter the file name as CM2102 date initials

10.   Type “lock”

11.   Select CDCl₃ as solvent

12.   Wait for “lock ready” and start shimming

13.   Select Z₁ – left or right. Click until the signal is at the highest level.

14.   Select Z₂ – left or right. Click until the signal is at the highest level.

15.   Repeat steps 14 and 15 until the signal is at the highest level, then press STDBY

16.   Type “rga”

17.   When “rga” is ready, type “zgefp”

18.   While waiting, type “setti” to set title for spectrum

19.   Type the title as CM2102 sample 7

20.   Wait until the spectrum is out

21.   Type “apk” to do phase correction

22.   Zoom into the region of the CDCl₃ (  ̴ 7 ppm)

23.   Calibrate the CDCl₃ peak at 7.26 ppm

24.   Click “Lift-On/Off” button to lift up the sample

25.   Remove the sample from the spectrometer

Results and calculations

(The effects of the chemical environments on the chemical shifts will be explored under the section, “Discussion”.)(For singlet, doublet and triplet coupling patterns, the chemical shift values given in this report will be their midvalues. For multiplets, the chemical shift values will be given as a range.)

For a molecule of ethyl acetate

Proton	Expected chemical shift, ppm	Observed chemical shift, ppm	Expected Coupling Pattern	Observed Coupling Pattern	Integral
Ha	0.7 - 1.3	1.2527	Triplet	Triplet	3
Hb	3.5 -4.8	4.0938 to 4.1364	Quartet	Quartet	2
HC	2 - 2.2	2.0382	Singlet	Singlet	3

Table 1: analysis of ethyl acetate

For a molecule of p-tolualdehyde

Proton	Expected chemical shift, ppm	Observed chemical shift, ppm	Expected Coupling Pattern	Observed Coupling Pattern	Integral
Ha	9.0 - 10.0	9.9595	Singlet	Singlet	1
Hb	6.5 – 8.0	7.7732	Doublet	Doublet	2
HC	6.5 - 8.0	7.3283	Doublet	Doublet	2
Hd	2.3 – 2.7	2.4376	Singlet	Singlet	3

Table 2: analysis of p-tolualdehyde

For a molecule of cyclohexene

Proton	Expected chemical shift, ppm	Observed chemical shift, ppm	Expected Coupling Pattern	Observed Coupling Pattern	Integral
Ha	1.2 – 1.4	1.5944 to 1.6191	Triplet	Multiplet	4
Hb	1.6 - 2.6	1.9800 to 1.9869	Doublet of triplet	Multiplet	4
HC	4.5 – 6.5	5.6652	Triplet	Singlet	2

Table 3: analysis of cyclohexenes

To determine the relative concentration of ethyl acetate, p-tolualdehyde and cyclohexene, distinct peaks of each molecule were identified. Distinct peaks are individual peaks of ethyl acetate, p-tolualdehyde and cyclohexene that do not overlap at the same chemical shifts. 

The distinct peaks are summarised below.

The peak at = 4.0938 to 4.1364 ppm belongs to ethyl acetate (H_b).
The peak at  = 5.6652 ppm belongs to cyclohexene (H_c).
The peak at   = 9.9595 ppm belongs to p-tolualdehyde (H_a).

	Hb of ethyl acetate	Ha of p-tolualdehyde	Hc of cyclohexene
No. of H per molecule	2	1	2
Approximate integral ratio at corresponding chemical shift	2	1	2
Relative concentration of molecule present	2/2 = 1	1/1 = 1	2/2 = 1

Table 4: determine the concentration ratio

Relative concentration ratio of ethyl acetate : p-tolualdehyde: cyclohexene = 1: 1 : 1.

To further confirm the relative concentration ratio, the calculations were repeated for the other peaks of the compounds.

	ethyl acetate		p-tolualdehyde			cyclohexene
	Ha	Hc	Hb	Hc	Hd	Ha	Hb
No. of H per molecule	3	3	2	2	3	4	4
Approximate integral ratio at corresponding chemical shift	3	3	2	2	3	4	4
Relative concentration of molecule present	1	1	1	1	1	1	1

Discussion

Nuclear Magnetic Resonance (NMR) spectroscopy makes use of the magnetic properties of certain nuclei such as proton to obtain information regarding the nature of the immediate environment of the magnetically distinct atom. NMR spectroscopy is the most important and widely used technique by chemists for identifying the structures of organic and inorganic compounds because it allows a full analysis of the entire spectrum for a compound.

In this experiment, the ¹H NMR spectrum was obtained to determine the concentration ratio of the three compounds in the mixture. Depending on its chemical environment, the protons in a molecule resonate at slightly different frequencies. The variation of the resonance frequency with chemical environment is known as the chemical shift. All protons in chemically identical environments within a molecule are chemically equivalent and exhibit the same chemical shift. The number of peaks corresponds to the number of chemically distinct types of protons in the molecule. The area under each peak is proportional to the number of hydrogens generating that peak. The protons in different magnetic environments of the same molecule can interact with one another through spin-spin coupling. This leads to the splitting of signals. The splitting pattern of a nucleus can be predicted by the (N+1) rule which states that a nucleus is split by N equivalent nuclei into (N+1) peaks. The ratio of line intensities within an NMR signal is determined by the Pascal’s triangle.

Solvent used

Deuterated solvent CDCl₃ was used to avoid the resonance from protons in the solvent from swamping the proton spectrum of compound. CDCl₃ was chosen because most compounds dissolve in it, it is also volatile so can be removed easily and it is inert thus no exchange of its deuterium with protons in the molecule being studied is possible. In addition, CDCl₃ can be used as a reference using the weak peak at 7.26ppm to ensure that the frequencies generated throughout the experiment remain constant. A small peak was observed for CDCl₃ in the proton NMR spectrum although deuterium is NMR inactive because there is a small percentage of CHCl₃ that is not deuterated.

Ethyl acetate

Ethyl acetate has an ester functional group. There are 3 different proton environments for ethyl acetate. Both H_b and H_c are more deshielded than H_a because they are closer to the electronegative oxygen atoms. The electron density on hydrogen is reduced by the electron withdrawing effect of oxygenleads to a reduction in local diamagnetic shielding that translates into an increase in deshielding and hence an increase in the chemical shift. The quartet signal, 4.0938 to 4.1364 ppm, is due to H_b coupling with 3 neighbouring H_a and it is the distinct peak for ethyl acetate as there are no other peaks in the region to overlap at the same chemical shift. The methyl group from H_a couples with 2 H_b to give a triplet at 1.2527 ppm. H_c is deshielded by the anisotropy field of the carbonyl groups to give a singlet at 2.0382 ppm as there are no neighbouring protons within 3 bonds.

p-tolualdehyde

p-tolualdehyde has a benzene ring and a carbonyl group with 4 proton environments. Hydrogens attached to benzene ring H_b and H_c are deshielded by the significant anisotropic field generated by the circulation of the π electrons in the ring.  Both H_batoms are chemically and magnetically equivalent as they are related by symmetry operation and the coupling with other protons is the same. H_b couples with H_c to form a doublet at 7.7732 ppm. As H_c is more distant from the electron-withdrawing carbonyl group than H_b, H_c experiences a smaller chemical shift at 7.3283 ppm. H_d is a benzylic hydrogen deshielded by the anisotropic field of the ring but they are further away from the ring, thus the effect is smaller. A singlet is observed at 2.4376ppm as there is no neighbouring ptotons within 3 bonds to couple with.

The chemical shift of the proton in the aldehyde group, H_a is at 9.9595 ppm. The high chemical shift is due to the anisotropy effect of the carbonyl group, the inductive effect of the electronegative oxygen decreases the electron density on the attached proton. Furthermore, H_a is bondedwith a sp² hybridized carbon thus it has more s-character than a sp³ hybridized carbon. sp² carbon atom holds electrons closer to the nucleus than sp³ carbon, this results in less shielding for the H nucleus. Thus the anisotropy effect and hybridization affect deshielded H_a therefore its chemical shift is towards a downfield region. A singlet is observed on this peak because no coupling occurred between its nearest neighbouring proton (H_b) as they are 4 bonds apart. This peak is indicative of an aldehyde group since no other protons appear in this region. An integral value of 1 is expected since there is only one of such proton in the compound.

Cyclohexene

Cyclohexene has an alkene functional group and a plane of symmetry that is perpendicular to the double bond, thus there are 3 different proton environments. An alkene has an area of low electron density in the plane of the molecule because the π orbital has a node there and the hydrogen nuclei lying in the plane gain no shielding from the π electrons. The hydrogens attached to the double bond are deshielded due to the anisotropic field of the π electrons in the double bond. The more distant the hydrogen from the double bond, the smaller the deshielding effect, therefore the allylic hydrogen H_b has smaller chemical shifts than H_c. When cyclohexene adopt the most stable conformation, half-chair, H_c and H_a can have long range coupling. Therefore, H_a has a higher chemical shift than expected from an ordinary CH₂ group. However, the interaction is almost negligible. H_a is expected to be a triplet as there are 2 H_bfor it to couple with. If a higher resolution NMR spectroscopy is available, H_b is expected to be a doublet of triplets because it can couple with 2 H_a and a H_c. But because of overlap with the strong peak from H_c of ethylacetate which has similar chemical shift, the pattern could not be distinguished easily. The signal appears as a multiplet. This problem can also be solved by recording the NMR spectrum again but at a different magnetic field. The distinct peak for cyclohexene is a singlet at 5.6652 ppm due to H_c. As there are 2 H_c in each molecule of cyclohexene, the expected integral is 2. For H_c, a triplet was expected due to coupling with 2 H_b, but instead a singlet was observed. This could be because when cyclohexene has a half chair or twisted boatconformation, H_bmay be oriented in away from H_csuch a way that there is minimal spin spin coupling.

Determine the concentration ratio

The NMR spectrum of a mixture of ethyl acetate, p-tolualdehyde and cyclohexene is a result of the superimposed spectrum of their individual spectrums. The distinct peaks selected are 9.9595 ppm, 5.6652 ppm and 4.0938– 4.1364ppm which belong to p-tolualdehyde, cyclohexene and ethyl acetate respectively. Using the experimentally determined integration ratio of the distinct peaks, the relative concentration of p-tolualdehyde, cyclohexene and ethyl acetate can be determined. The results were then double checked with the integration ratios observed from other peaks.

Conclusion

Proton NMR of a liquid mixture consisting of ethyl acetate, p-tolualdehyde and cyclohexene in CDCl₃ solvent was obtained and the peaks were assigned. The relative concentration ratio of ethyl acetate p-tolualdehyde and cyclohexene was found to be 1: 1 : 1 based on integration of the distinct peaks.

References

1)      Donald L. Pavia, Introduction to Spectroscopy, Brooks/Cole, Cengage Learning

2)      Alan K. Brisdon, Inorganic Spectroscopic Methods, Oxford Science publications

3)      Inorganic Chemistry 4^th Edition, Shriver & Atkins, Oxford University Press

4)      Banwell and McCash, Fundamentals of Molecular Spectroscopy, 4^th edition.

5)      http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/nmr/nmr1.htm

[accessed 10/03/11]

6)      http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/nmr1.htm

[accessed 11/03/11]