Lab Report on the NMR Spectroscopy of Organic Molecule

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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 (Hb).
The peak at
 = 5.6652 ppm belongs to cyclohexene (Hc).
The peak at
  = 9.9595 ppm belongs to p-tolualdehyde (Ha).

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 Hb and Hc are more deshielded than Ha 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 Hb coupling with 3 neighbouring Ha 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 Ha couples with 2 Hb to give a triplet at 1.2527 ppm. Hc 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 Hb and Hc are deshielded by the significant anisotropic field generated by the circulation of the π electrons in the ring.  Both Hbatoms are chemically and magnetically equivalent as they are related by symmetry operation and the coupling with other protons is the same. Hb couples with Hc to form a doublet at 7.7732 ppm. As Hc is more distant from the electron-withdrawing carbonyl group than Hb, Hc experiences a smaller chemical shift at 7.3283 ppm. Hd 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, Ha 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, Ha 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 Ha 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 (Hb) 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 Hb has smaller chemical shifts than Hc. When cyclohexene adopt the most stable conformation, half-chair, Hc and Ha can have long range coupling. Therefore, Ha has a higher chemical shift than expected from an ordinary CH2 group. However, the interaction is almost negligible. Ha is expected to be a triplet as there are 2 Hbfor it to couple with. If a higher resolution NMR spectroscopy is available, Hb is expected to be a doublet of triplets because it can couple with 2 Ha and a Hc. But because of overlap with the strong peak from Hc 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 Hc. As there are 2 Hc in each molecule of cyclohexene, the expected integral is 2. For Hc, a triplet was expected due to coupling with 2 Hb, but instead a singlet was observed. This could be because when cyclohexene has a half chair or twisted boatconformation, Hbmay be oriented in away from Hcsuch 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 4th Edition, Shriver & Atkins, Oxford University Press
4)      Banwell and McCash, Fundamentals of Molecular Spectroscopy, 4th edition.
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