NMR Spectroscopy in the Undergraduate Curriculum - ACS Publications

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Measurement of Phosphates in Soft Drinks: A General Chemistry Experiment Using NMR Laura Medhurst,* Farzana Shahnaz, Nirmala Ramnarine, and Giordano Paniconi Department of Chemistry, Marymount University, Arlington, Virginia 22207, United States *E-mail: [email protected]

NMR is most commonly introduced in the organic chemistry curriculum. This experiment is an introduction to NMR spectroscopy specifically designed for introductory chemistry students. No prior knowledge of chemical shift or spin-spin splitting is required. Students compare a standard curve prepared from the integrated peak area of phosphate solutions, with known concentrations, to the integrated peak area of the phosphate peak of various soft drinks.

Introduction Marymount University is a comprehensive Catholic university in Arlington, Virginia. In January 2011, a new science facility, Caruthers Hall, opened. In Caruthers Hall, the combined biology, chemistry and physics laboratory space more than doubled, and the combined science department secured dedicated research facilities. In the past four years the number of biology majors has increased by 3.0% per year, and a new biochemistry major has been implemented, which includes an expanded chemistry curriculum. In conjunction with construction of the new building, Marymount University received $200,000 through the U.S. Department of Education Fund for Improvement of Postsecondary Education (FIPSE) grant program in 2010, "for science equipment and technology." This grant was used to purchase a Bruker 400 MHz Ascend NMR spectrometer. This equipment was chosen because of the emphasis on NMR spectroscopy in the American Chemical Society in its Guidelines for Accreditation of Undergraduate Programs, which states, “Approved programs © 2016 American Chemical Society

Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: First Year and Organic Chemistry Courses Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


must have a functioning NMR spectrometer that undergraduates use in instruction and research. The Committee strongly recommends an FT-NMR spectrometer” (1). To modernize our laboratory program, we decided to increase the use of advanced instrumentation in even our introductory laboratory courses. Previously we had included Fourier Transform IR in our introductory courses, but NMR spectroscopy presented more difficulties. Most of the published experiments using NMR were for organic chemistry courses or upper division courses (2). NMR is traditionally introduced in organic chemistry, and we wanted to develop an experiment, which would not require knowledge of organic chemistry, and would not require knowledge of even the elementary properties of NMR such as chemical shift or spin-spin splitting, in order to leave these rather difficult concepts in the organic chemistry curriculum. This makes the emphasis of the lab on the parts of the instrument, the method of recording an NMR spectrum, and processing the spectrum. Two other criteria, which are important for any general chemistry experiment, which uses shared instrumentation, are the experiment should be relatively easy for adjunct faculty to supervise and perform, and all the students in a lab section, a maximum of sixteen, must be able to take and analyze at least one spectrum during the three hour lab. One published experiment looked probable with respect to these criteria. This experiment used 31P NMR to characterize the pH dependence of phosphates (3). In this experiment, phosphate buffers are made with different pH. The NMR is used to measure the chemical shift of 31P as a function of pH. Typical data are shown in Figure 1.

Figure 1. Chemical shift of 31P as a function of pH. There is only one peak in the NMR spectrum of phosphate buffers because interconversion between the two major phosphate ions is rapid compared to the time of NMR relaxation. The chemical shift of the one phosphate peak depends on the relative concentrations of the two dominant species, H2PO4- and HPO42-. 32 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: First Year and Organic Chemistry Courses Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Although it initially seemed this experiment could be used to introduce both the NMR instrument and titration curves, this experiment was ultimately rejected as an introductory lab, because the students are just beginning to learn how pH affects the concentration of a weak acid and its conjugate base, and we would need to introduce them to chemical shift and the relative rates of the NMR relaxation and hydrogen ion transfer to complete the picture. This experiment was deemed much more appropriate for students who had already taken organic chemistry. Ultimately we modified a visible absorption experiment, which uses Beer’s Law to determine the concentration of phosphorous in stream water (4). In the stream water experiment, phosphorous in the phosphate form reacts with ammonium molybdate to form ((NH4)3[PMo12O40]), which is reduced by SnCl2 to form an intensely blue compound, the β-keggin ion, ([PMo12O40]7-) (5). The amount of the blue colored ion produced is proportional to the amount of phosphorous present. This technique is very sensitive and can detect phosphorous in the sub-ppm level. The absorption can be measured using a colorimeter with a maximum absorbance at 720 nm to determine the amount of phosphorus.

Experimental Details A Bruker Ascend 400 MHz spectrometer was used to collect the data. This instrument is controlled by the Topspin software package. The pulse program used was ZG-30, which is a simple one-pulse sequence. Initially the students compiled 32 scans, however we increased this to 128 to improve the signal to noise ratio. Carbonation was removed from the soda samples by first heating and then cooling a 50 mL sample of the soda. A 0.5 mL sample of soft drink was then added directly in the NMR tubes and combined with 0.2 mL of D2O. Although all of the phosphorous present is in a phosphate, it is present as both H2PO4- and HPO42-. Because the relative amounts of these ions are a function of the pH, and not all of the samples have the same pH, the concentration is reported as parts per million of phosphorous. The primary phosphorous standard was made by dissolving 1.098 g of anhydrous KH2PO4 in 1.000 liter of purified water. This solution has a concentration of 250 ppm of phosphorous. The other standards were made by dilution of the primary standard. Each standard (0.50 mL) was also combined in the NMR sample tube with 0.20 mL of D2O. To save time, the standards were analyzed first by the lab instructors, and the results were given to the class. The lab instructions contain detailed directions for the collection of a spectrum using the Bruker 400 MHz Ascend NMR spectrometer, and a description of why each step is necessary. Each student selected one soda with an unknown phosphorous concentration and prepared the sample. They then performed all of the steps necessary to record a spectrum, including creating a new data file, removing the previous sample from the instrument, inserting a new sample into the spectrometer, locking the solvent signal, tuning the probe to sample, shimming the magnetic field, and setting the gain. A sample of typical free induction decay (FID) is shown in Figure 2.

33 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: First Year and Organic Chemistry Courses Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 2. Free Induction Decay 250ppm phosphorous.

The students then process the FID and apply an automated phase adjustment to the spectra. Figure 3 shows a processed spectrum of the 250 ppm standard after 32 scans and also after 128 scans. We determined the improvement in the signal to noise justified the extra three minutes of acquisition time for each spectrum.

Figure 3. Comparison of processed spectrum after 32 scans with spectrum after 128 scans. 34 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: First Year and Organic Chemistry Courses Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


If the automatic peak-picking and integration functions are used, the software typically assigns noise in the spectrum as peaks, because the signal to noise ratio is poor for this experiment, especially for the less concentrated samples. Therefore it is easier for the students to manually pick the single peak and integrate it. Because the pH values of the samples are all similar, the students simply look for a peak close to 0.0 ppm. The students construct a Beer’s Law plot from the integrated peak area of the standards. Typical results are shown in Figure 4.

Figure 4. Concentration of phosphorous in ppm versus integrated peak intensity.

Each integrated peak area for the soft drinks was compared with the standard curve. The values for phosphorous concentration are shown in Table 1.

Table 1. Concentration of Phosphorous in Soft Drinks Using NMR Soft drink

Phosphorous concentration (ppm)

Sprite

43

Pellegrino Sparkling Water

0

Coca Cola

148

Caffeine-Free Pepsi

147

Gatorade

77

Vitamin Water

370



These results compare well with the known concentration of phosphorous in sodas of 155 ppm for dark colas (Pepsi and Coca Cola) and the absence of it, based on the list of ingredients, in others (Sprite and Pellegrino) (6). We introduced this experiment in the spring semester of 2014. In the conclusion section of their reports, the students mainly emphasized that the integration of the peak area was proportional to the amount of the nuclei tested. One possible modification of this experiment would be to use an internal standard by adding methylphosphonic acid as a 31P-NMR standard to the NMR samples (7).In this case, the chemical shift must be introduced to explain the two phosphorous peaks, which caused us to reject this modification. To keep the majority of the students occupied productively while the other students were running their NMR spectra, a second part was added to this experiment in the subsequent semesters. The UV/VIS technique previously discussed was added to the lab to also include a comparison with the NMR. A similar experiment using visible spectroscopy, but with a different chromophore, has been previously described (8). This technique is much more sensitive than NMR, so many of the soft drink samples must be diluted before they may be tested using this technique. The students are required to perform a 1:10 and a 1:100 dilution of the soda with reverse osmosis water. The students prepare their own standard curve with a maximum of 2.5 ppm concentration and compare the absorbance of the undiluted sample, and the two diluted samples with this standard curve. The values of phosphorous for the same unknown samples using this technique are given in Table 2.

Table 2. Concentration of Phosphorous in Soft Drinks Using UV/VIS Soft drink

Phosphorous concentration (ppm)

Sprite

0

Pellegrino Sparkling Water

0

Coca Cola

126

Caffeine-Free Pepsi

66

Gatorade

53

Vitamin Water

150

The data consistently show that this optical technique tends to give a concentration below that found using NMR, and the NMR results were more consistent with the previous results (7, 8).


Conclusions


Although the experiment we developed is quite simple, it accomplished several of our instructional goals. The introductory chemistry students were given an opportunity to use a sophisticated instrument and even to prepare and run their own samples. The spectra were easily analyzed by the students, who were familiar with the concept of the concentration of the sample being proportional to intensity of the sample’s absorbance of visible light. By including the comparison with the UV/VIS technique, this experiment also illustrates some fundamental principles of analytical chemistry. Most measurements can be accomplished by more than one method, but for a given sample one method may be superior to another.

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Undergraduate Professional Education in Chemistry: ACS Guidelines and Evaluation Procedure for Bachelor’s Degree Programs. American Chemical Society. http://www.acs.org/content/dam/acsorg/about/ governance/committees/training/2015-acs-guidelines-for-bachelors-degreeprograms.pdf (accessed July 16, 2015). NMR Spectroscopy in the Undergraduate Curriculum; Soulsby, D, Anna, L. J., Wallner, A. S. Eds.; ACS Symposium Series 1128; American Chemical Society: Washington, DC, 2013. Jabbour, J., D. Dolino, S., S. Simmons, S., Steiger, M. A., Malloy, T. B. Raman and 31P NMR Characterization of the pH Dependence of Aqueous Phosphates: An Undergraduate Experiment. In Abstracts of Papers, 235th ACS National Meeting, New Orleans2008. Standard Methods for the Examination of Water and Waste Water; Greenberg, A. E., Trussel, R. R., Clesceri, L. S., Franson, M. A. H., Eds.; American Public Health Association, American Water Works Association, Water Pollution Control Federation: Washington, DC, 1985; pp 446−448 Barrows, J. N.; Jameson, G. B.; Pope, M. T. Structure of a Heteropoly Blue. The Four Electron Reduced beta-12-Molybdophosphate Anion. J. Am. Chem. Soc. 1985, 107, 1771–1773. Phosphorus and Your CKD Diet. National Kidney Foundation. https://www.kidney.org/atoz/content/phosphorus (accessed July 29, 2015). Kollo, M.; Kudrjasova, M.; Kulp, M.; Aav, R. Methylphosphonic Acid as a 31P-NMR Standard for the Quantitative Determination of Phosphorus in Carbonated Beverages. Anal. Meth. 2013, 5, 4005–4009. Murphy, J. Determination of Phosphoric Acid in Cola Beverages. J. Chem. Educ. 1983, 60, 420–421.


NMR Spectroscopy in the Undergraduate Curriculum - ACS Publications

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