The effect of recrystallization can also be observed by comparing the spectra of the crude and the final product. In the spectra of the crude product, the appearance of a resonance up-field from the methyl is observed (1.99 ppm, marked with a star). This is acetic acid that has not been completely removed during work-up. Next to this peak (at 2.02 ppm) traces of methyl acetate form an esterification between acetic acid and d-methanol can be seen. In addition, at the methyl resonance of ASA, minor resonances appear and overlaps with the methyl signal. This is not due to contamination of the reagents or solvents proven by the fact that the 1H-NMR spectra of all reagents and solvents were pure. It might be caused of side-reactions, such as formation of dimers or polymers with methyl groups resonating in this area. However, these contaminants, and thus the overlapping signals, disappears after recrystallization.
Minor resonances at 1.13 and 3.26 ppm in the spectra of acetic acid anhydride are assumed to be satellites of the methyl signal, which can be in consistence with the observation of a satellite of the methyl group in ASA with the resonance at 1.21 ppm in both the spectra of the crude and final product of ASA. The other satellite is probably overlapping with the methanol resonance. However, this resonance is so small that it can be neglected in the calculation of the yield.
4. Discussion and Concluding Remarks
The students handled the synthesis and 1H-NMR analysis very well with the necessary guidance. Furthermore, they expressed that they found it motivating to analyze their own product. Since benchtop 1H-NMR will most probably be extensively used in the future, also in medical laboratories, it is important that undergraduate students get an introduction to a simplified 1H-NMR theory and procedure to use the instrument. Further, it is more motivating to get hands on experience with acquiring and interpreting spectra, as opposed to a theoretical approach in a regular classroom setting. The experiment allowed the students to become proficient operator of a benchtop 1H-NMR. They understood the basic experimental parameters involved in the acquisition of the 1H-NMR spectra, and they also gained experience with processing spectra and data analysis using 1H-NMR software.
This experiment, synthesis and NMR analysis can relatively easily be implemented in High Schools and for undergraduate students, and by teachers/instructors. In addition, the students get a nice and informative overview of the possibilities and the advantages of the NMR technique. This work shows that an introduction to one of the most demanding and advanced methods in Chemistry can be introduced at an early stage in the curriculum of Chemistry to promote the chemistry career. The assignment is based on self-study, and with helped by this article and the Supporting Material, the time necessary for the instructor is minimized. This is normally the threshold of a laboratory experiment when advanced instrumentation is used.
Further Work
To evaluate the laboratory exercise, both the synthesis and application of 1H-NMR, it will be useful to make a survey on several student groups. Further, to enhance learning, it would be valuable for students to produce videos were both the synthesis and the application of the 1H-NMR instrument are demonstrated 23. In addition, to reduce the time spent supervising by instructors and technicians during the exercise, it would have been useful to make videos of 1H-NMR theory that covers the theory the students need to perform the exercise.
Supporting Material
1. Experimental. The synthesis of Acetylsalicylic acid
This is an experimental procedure based on an article in the Journal of a Chemical Education, 1998, by John Olmsted. 24
Equipment:
Erlenmeyer flask (125 mL), graduated cylinders (5 mL and 20 mL), spatula, weighing boats, rubber stopper with a ventilating needle, syringe (2 mL), water bath, filter papers, Hirsh funnel fitted to a vacuum flask, glass rods, wiper, beakers (2x 50 mL), a heating plate and an ice bath.
Chemicals:
– Salicylic acid
– Acetic acid anhydride
– Phosphoric acid (85% water)
-d-methanol 99% added 0.03% TMS
Checking the quality of the reagents:
1) Perform an 1H-NMR analysis of the reagents, following the procedure described in chapter 2, to ensure that they are intact before the synthetic experimental work begins.
The synthesis:
2) In an Erlenmeyer flask (125 mL), add salicylic acid (1.4 g), acetic acid anhydride (3.0 mL) and 5 drops of the catalyst, phosphoric acid.
3) Fit a rubber stopper containing a ventilating needle to the top of the Erlenmeyer flask.
4) Swirl the flask with the reaction mixture in a pre-heated water bath (90 °C) till the starting material is dissolved. Then fit the flask to a rack and leave it in the bath for 5 minutes.
5) Remove the flask from the water bath and carefully add 2 mL deionized water through the stopper with a syringe. Water facilitates product formation and decomposes the remaining acetic acid anhydride, both of which are exothermic reactions.
6) When the flask is sufficiently cold, remove the rubber tubing, and add another 20 mL deionized water.
7) After the product mixture has reached room temperature, and precipitation has started, further add 10 mL deionized water, and place the flask in an ice bath for 1 hour.
Vacuum filtration:
8) Collect the crystals by vacuum filtration on a filter paper fitted in a Hirsh funnel. Rinse the Erlenmeyer flask with 15 mL deionized water and pour it into the Hirsh funnel. A wiper fitted to a glass rod should be used to collect most of the remaining crystals on the glassware.
9) Continue vacuum suction for 10 minutes.
10) Transfer the crystals to a pre-weighed beaker (50 mL) and weigh the crude product.
11) A small quantity (ca 10 mg) of the crude product should be saved for 1H-NMR analysis.
Recrystallization:
12) Add 10 mL deionized water, per gram product, to the beaker.
13) Heat the solution to 85-90 °C on a heating plate whilst stirring with a glass rod till all product is dissolved.
14) Then leave the beaker on the bench to slowly reach room temperature before placing it in an ice bath. Slow cooling optimizes for crystal growth.
15) Collect the recrystallized product by vacuum filtration as described above (8-10).
16) Place the beaker with the final product in an oven at 80 °C for at least 1 hour, before it is cooled, weighed, and stored in a sealed container at 4 °C for further analysis by 1H-NMR.
17) Perform an NMR analysis of the crude product and final product following the procedure described in chapter 2.
18) Describe what you observe in all the recorded spectra and compare your result with Table 1 in chapter 2.
19) Calculate the yield by method A and B, as described in chapter 3, and comment on the result.
2. 1H-NMR analysis. Sample preparation and settings of 1H-NMR parameters.
The spectra in this article were acquired on a Margitek Spinsolve 60 MHz instrument equipped with MestReNova software. The samples were added to Norell NMR sample tubes and dissolved in 0.750 ml d-methanol (99%) added 0.03% TMS provided from ChemSupport AS. The 1H-NMR spectra were recorded after running a quick shim to homogenize the magnetic field. All the 1H-NMR spectra were recorded with 32 transients, a repetition time of 10 seconds, pulse angle of 90o and with an acquisition time of 6.4 seconds. The recorded spectra were saved in MestReNova for further data processing.
Spectra collected in the laboratory exercise.
a) d-methanol (or other deuterated solvent).
b) Salicylic acid.
c) Acetic acid anhydride.
d) Phosphoric acid (85% water)
e) Crude product; acetylsalicylicacid.
f) Final product; acetylsalicylicacid.
Data processing.
The spectra were processed with automatic phasing and baseline correction. The reference (TMS) where set to a chemical shift of 0 ppm and the resonances in the spectra were adjusted accordingly. The integral of the methyl resonance and of the aromatic region were manually integrated in MestReNova. The methyl peak was set to 3.00 corresponding to the three hydrogens of the group.
Interpreting the NMR spectra:
The NMR spectra of the reagents gives us information on whether there are any contaminants present and makes it possible to observe disappearing or appearing resonances during the synthesis. The results from the 1H-NMR spectra of the compounds dissolved in d-methanol are listed in Table S1. The recorded spectra are found in chapter 4.
3. Calculations
Method A: The yield of the reaction is found by using equation 1, in Table 1 in the article, after the weight of the starting material (SA) and final product (ASA) has been converted to moles. Raw data is found in Table 2 in the article.
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Method B: The ratio of the integral form the water signal and the methyl group obtained from the 1H-NMR spectra of pure d-methanol (Figure 2 in chapter 4) was calculated to 4.11/3 = 1.37. Check your NMR solvent for the correct ratio. These signals also appear in the spectra of the final product, and if the ratio exceeds 1.37, water is contaminating the sample. The area of the water signal in the sample was corrected by formula 3, to eliminate water from the solvent.
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(3) |
Equation 4 is then solved with respect to moles of ASA, 
where the mole of water in the sample can be expressed by formula 5 and inserted into formula 4, where 
is the mass in gram and 
is the molecular weight.
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(4) |
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(5) |
When the integral of the methyl group is set to 3.00, the methyl in d-methanol gave an integral of 0.28. Based on the ratio (1.37) described above, the equation 2 in Table 1 in the manuscript and equations 3-5 is used to find the yield of the final product:
Area of the H2O resonance in d-methanol: 
Area of the H2O resonance in ASA:
(3)
(4 and 5)
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Yield method B:
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(2) |
4. The 1H-NMR-spectra used in the article.
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Figure S1. The 1H-NMR spectra of acetylsalicylicacid in d-chloroform
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Figure S2. The 1H-NMR spectra of d-methanol with 0.03% TMS
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Figure S3. The 1H-NMR spectra of salicylicacid in d-methanol with 0.03% TMS
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Figure S4. The 1H-NMR spectra of acetic acid anhydride in d-methanol with 0.03 % TMS. The cutout spectra show the area of 1-5 ppm and includes resonances from impurities, solvent, and satellites
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Figure S5. The 1H-NMR spectra of phosphoric acid (85%) in d-methanol with 0.03% TMS. No impurities were found
Published with license by Science and Education Publishing, Copyright © 2022 Marit Kristin Leiren and Signe Steinkopf
This work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit
http://creativecommons.org/licenses/by/4.0/Cite this article:
Normal Style
Marit Kristin Leiren, Signe Steinkopf. Introducing NMR to Biomedical Laboratory Scientists through a Laboratory Exercise; Synthesis, Structure Determination and Quantization of Aspirin by Employing an 1H-NMR Bench Top Instrument. World Journal of Chemical Education. Vol. 10, No. 1, 2022, pp 8-19. http://pubs.sciepub.com/wjce/10/1/2
MLA Style
Leiren, Marit Kristin, and Signe Steinkopf. “Introducing NMR to Biomedical Laboratory Scientists through a Laboratory Exercise; Synthesis, Structure Determination and Quantization of Aspirin by Employing an 1H-NMR Bench Top Instrument.” World Journal of Chemical Education 10.1 (2022): 8-19.
APA Style
Leiren, M. K. , & Steinkopf, S. (2022). Introducing NMR to Biomedical Laboratory Scientists through a Laboratory Exercise; Synthesis, Structure Determination and Quantization of Aspirin by Employing an 1H-NMR Bench Top Instrument. World Journal of Chemical Education, 10(1), 8-19.
Chicago Style
Leiren, Marit Kristin, and Signe Steinkopf. “Introducing NMR to Biomedical Laboratory Scientists through a Laboratory Exercise; Synthesis, Structure Determination and Quantization of Aspirin by Employing an 1H-NMR Bench Top Instrument.” World Journal of Chemical Education 10, no. 1 (2022): 8-19.
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- Figure 1. 1H-NMR of ASA in d-chloroform, recorded with a 60 MHz instrument from Magritek. The resonance at 11.89 ppm is from the acid proton, the resonance around 7.5 ppm are from the aromatic protons and the resonance at 2.43 is from the methyl group
- Figure 2. Spectrum 1 shows the 1H-NMR spectra of SA, with the combined resonance from the alcohol group and the water signal at 5.29 ppm, and the resonances that originate from the aromatic hydrogen atoms in the chemical shift region of 6.73-7.95 ppm. Spectrum 2 shows the 1H-NMR spectra of phosphoric acid catalyst (85% water) with the resonance from water at 5.73 ppm. Spectrum 3 shows the 1H-NMR spectra of acetic acid anhydride with a resonance at 2.19 ppm from the methyl groups present. Minor impurities at 2.02 and 1.98 ppm and satellite signals from the methyl group are observable in a cut-out spectra of acetic acid anhydride in Supporting Material (figure 4). Spectrum 4 shows the 1H-NMR spectrum of 99% d-methanol with TMS at 0.00 ppm. The resonance at 3.31 ppm originates from the non-deuterated methyl group and the resonance at 4.83 ppm is appearing from the alcohol group in methanol and water.
- Figure 3. Spectrum top to bottom, show the 1H-NMR spectra of SA (top), the crude product ASA (middle) and the final product, ASA (bottom). The cutout of the 1H-NMR spectra includes the regions where we find the methyl group (1.6-3.00 ppm) and the aromatic region (6.6-8.5 ppm)
- Figure 4. The 1H-NMR spectrum of the final product of ASA showing the resonances with integrals that were used for quantification of the yield. The integral of the methyl resonance (2.88 ppm) was set to 3.00 and the resonances in the aromatic chemical shift region showed 4.00. In addition, the area of the methyl group in methanol (3.31 ppm, 0.28) and the water signal (4.87 ppm, 1.10) are shown
- Figure S4. The 1H-NMR spectra of acetic acid anhydride in d-methanol with 0.03 % TMS. The cutout spectra show the area of 1-5 ppm and includes resonances from impurities, solvent, and satellites
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