NMR at the University of St. Thomas (TX): Cooperation and

Chapter 7

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NMR at the University of St. Thomas (TX): Cooperation and Collaboration with Rice University Thomas B. Malloy, Jr.,*,1 Michelle A. Steiger,1 and Lawrence B. Alemany2 1Department

of Chemistry & Physics, University of St. Thomas, Houston, Texas 77006, United States 2Department of Chemistry and Shared Equipment Authority, Rice University, Houston, Texas 77251, United States *E-mail: [email protected]

The University of St. Thomas (TX) is a private school with an undergraduate enrollment of approximately 1600 and a total enrollment of 3500. All STEM areas are strictly undergraduate. This chapter describes 1D- and 2D-NMR experiments, and applications of 1H, 13C, 31P and 19F NMR specroscopy with the Anasazi Eft 60 in both laboratory courses and undergraduate research. Access to high field instrumentation was obtained through cooperation of nearby Rice University and research on complex splitting patterns in simple organofluorine compounds is described.

Introduction The Anasazi Eft 60 FT NMR was obtained through the National Science Foundation Course Curriculum and Laboratory Improvement program. For the University of St. Thomas (UST), this was the last hurdle in being able to apply for accreditation through the Committee on Professional Training, American Chemical Society for the programs in Chemistry and Biochemistry. Since there were no faculty members with an extensive background in NMR, an Advisory Committee from local, nearby facilities was formed. These included representatives from Sam Houston State University, the Shell Research Center in Houston, the University of Houston and Rice University. This committee © 2016 American Chemical Society Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


reviewed the proposal before it was submitted and made recommendations for improvement. Since then, they have provided examples of applications, and provided suggestions. The relationship with nearby Rice University became particularly close and has been mutually advantageous. One of our challenges was to make the most effective use of the instrument. The most obvious application of NMR in the undergraduate curriculum is in the sophomore organic class. For many years at UST, the first lab period in this course was an extended lecture on spectroscopic methods including infrared spectroscopy, mass spectrometry, UV-visible spectroscopy and NMR. This was accompanied by handouts with examples and exercises to be completed by the students over a period of time. The variety of experiments we could perform in this class with 80+ students was limited and required coordination among the other courses using the instrument. A number of excellent examples of applications to organic chemistry have been given in Volume 1 (1–6) of this series. Our use in the organic course has been limited to running proton spectra of pure compounds and reaction mixtures to determine reaction completion and as an exercise in identifying unknowns by applying a combination of wet chemical and instrumental techniques. 13C spectra are run as single examples for a given lab group. A kinetics experiment on basecatalyzed deuterium exchange of ketones is a complement to a GC/MS experiment performed by the first semester organic lab students. The rest of this chapter describes UST’s efforts to introduce 1D- and 2D-NMR to students at every undergraduate level and to introduce selected second semester freshmen to NMR spectroscopy, including chemical shifts, first-order spin-spin splitting, effects of time averaging and proton exchange, proton counting, quantitative applications and identification of unknowns. In the physical chemistry lab 31P spectra are applied to phosphate solutions at different pH values, compared to titrations and to Raman spectra of the same solutions. A combination of 1H spectra and 31P spectra, with and without proton decoupling of biologically important phosphorous molecules, along with spectral simulation are used to determine coupling constants. The biochemistry course includes 2D experiments on dipeptides including COSY and HETCOR experiments. Undergraduate research has played an important role in the integration of NMR into the curriculum and has led to the use of high field instruments at Rice University on an ad hoc basis for several projects when the data from the 60 MHz instrument were not sufficient. This led to an active collaboration over several years when hither unreported phemomena for complex patterns in simple organofluorine molecules were discovered.

NMR for First Year Students Chemical Shifts, Spin-Spin Splitting, and Proton Counting Freshman students are familiar with electronic structure, electron spin, the concepts of energy levels, absorption and emission of photons, frequencies and wavelengths. They also understand electronegativity and shielding effects. The introduction of the concepts of nuclear spin and chemical shifts were readily accepted. Since protons have the same familiar spin ½ as electrons, the notion of 94 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


energy level separation in a magnetic field was also readily accepted. Other spins were mentioned, but only as a matter of information. Based on performance in the first semester of General Chemistry, selected students were offered the opportunity to work on an NMR project. These were divided into groups of approximately 4 students, each group meeting one afternoon a week, for 4-6 weeks with upper level students acting as mentors and a faculty member supervising. The following is a composite of experiments used over several years. After an introductory lecture covering chemical shifts and demonstration in the lab, the students prepared model compounds for NMR analysis, obtained the spectra and analyzed them in networked computers in a separate room. The model compounds are alcohols, ketones and esters. Acetone, methanol and methyl acetate are used as examples to discuss functional groups, shielding and the quantitative response of NMR to the number of protons (7). For the sake of simplicity and speed, neat samples with the addition of a small amount of tetramethylsilane (TMS) were used. This also allowed instruction on handling low boiling materials at low temperatures in a hood. The spectra are shown in Figure 1. The follow up was to introduce spin-spin splitting from protons on adjacent carbons. The models used were 3-pentanone, ethanol and ethyl acetate. The diagram in Figure 2 was used to explain the splitting in an ethyl group. It was emphasized that the same coupling constant applied to the splitting of the methyl by the methylene group and the splitting of the methylene by the methyl group. The relation of the intensities to Pascal’s triangle was pointed out.

Figure 1. Proton NMR spectra of (a) acetone, (b) methanol, and (c) methyl acetate. In order to calculate the multiplicity, the equation 2nI+1 was used with n, the number of nuclei responsible for the splitting, and I the spin of the nucleus. There was some discussion about the definition of n, but after going back and forth, the idea that it is 2 for a methyl group adjacent to methylene and 3 for methylene adjacent to methyl, was accepted. Since I= ½ for protons, the equation normally found, n+1, was used but the students were urged to remember that it also applies when I≠ ½. 95 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 2. Spin-spin splitting pattern from protons on adjacent carbon atoms in an ethyl group. At that point, we obtained spectra of 3-pentanone, ethyl acetate and ethanol. The spectra of the first two are given in Figure 3 where assignments are indicated in the figure along with the integrals. By comparison with the spectra in Figure 1, we were able to discuss similarities and differences in chemical shifts, the calculation of multiplicity, the relation of the integrals to the number of protons and, qualitatively, the distribution of intensity within a multiplet.

Figure 3. Proton NMR spectra of (a) 3-pentanone and (b) ethyl acetate. For convenience, we had chosen to run the samples neat with a small amount of TMS. The use of absolute alcohol was fortuitous. First of all, with the OH proton split by the methylene group into a triplet, and the CH2 quartet additionally being split by the single OH proton it was possible to illustrate the stepwise application of the n+1 (2nI+1) calculation of multiplicity for the CH2 by non-equivalent groups of protons (Figure 4a). We had another fortuitous occurrence. A small amount of absolute alcohol was left in an open container for two days in the humid Houston air. This turned out to give us an additional opportunity to discuss another phenomenon, namely the NMR time scale and exchange reactions.

96 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 4 shows the NMR spectra of (a) absolute ethanol, (b) ethanol with water absorbed from the air, (c) ethanol with a small amount of deionized water (slightly acidic) added, and (d) ethanol to which a drop a HCl was added. The transition of the OH from a well-defined triplet then broadened and finally to a sharp singlet is seen. The CH2 goes from an overlapped quartet of doublets, broadens and then becomes a sharp quartet.

Figure 4. NMR spectrum of ethanol (a) absolute, (b) exposed to wet air, (c) DI water added, (d) dilute HCl added.

Quantitative Applications At this point, the students had been exposed to nuclear spin, chemical shift, shielding, proton counting, spin-spin splitting and multiplicity. We then applied the quantitative properties of NMR to several systems. In a given year, the different groups did the same experiments and compared or pooled results. The first of these was the measurement of ethylene glycol water mixtures (8). Another fortuitous accident occurred. A bottle of ethylene glycol from the stockroom was used by the faculty member to attempt to demonstrate the 2:1 area ratio between the CH2 and OH protons. The result was not even close. The lid was loose or left off for a considerable time and water absorbed by the air. A second bottle yielded the correct result shown below in Figure 5. This allowed the students to observe the power of experimental science to resolve an anomalous result.

Figure 5. Proton NMR spectrum of ethylene glycol. 97 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Solutions of ethylene glycol and water were made up by volume to illustrate the quantitative nature of NMR Spectroscopy. Assuming ideal solution behavior, volume % was converted to mass % and mole % using densities and molar masses. Measurements were made on solutions and ½ CH2 area subtracted from the OH peak to yield the amount from water (Figure 6). This allowed calculation of mole, mass and volume % based on NMR given in Table 1.

Figure 6. Integrated NMR spectra of ethylene glycol (EG) containing samples.

Table 1. MonoEthylene Glycol (MEG) - Water Solutions Mole % MEG

Mole % H2O

Vol % MEG

Calculated

NMR

Calculated

NMR

Vol % H2O

25%

9.7%

9.9%

90.3%

90.1%

75%

50%

24.5%

25.7%

75.6%

74.3%

50%

75%

49.3%

47.2%

50.7%

52.8%

25%

100%

100%

100%

0%

0%

0%

The above was applied to three samples. The first was the bottle of ethylene glycol that had obviously absorbed water. The other two were a new, unopened bottle of antifreeze and a closed partial bottle with a small amount of unused antifreeze. The mole %, mass % and volume % for the 3 samples are given in Table 2 . In practical terms, volume % was probably of the most interest. It was comforting that the unopened antifreeze had only 2.1% water and appalling that the MEG for the stockroom had over 15% water. Results from a similar experiment measuring the alcohol content for whiskey and wine samples are given in Figure 7 and Table 3. The results on the whiskey and wine were the average values from eight students. The relative % error from the values on the labels are also given in Table 3. 98 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Table 2. NMR Analysis of MEG and Antifreeze Samples


water

Ethylene glycol

Vol %

Mass %

Mol %

Vol %

Mass %

Mol %

MEG sample

15.8

14.4

36.7

84.2

85.6

63.3

Unopened antifreeze

2.1

1.9

6.1

97.9

98.1

93.9

Opened antifreeze

21.7

19.9

46.1

78.3

80.1

53.9

Figure 7. NMR spectra of whiskey (80 proof) and red wine (13.5%).

Table 3. NMR Determination of Alcohol Content NMR Results

Label

Mol %

Mass %

Vol %

STD Dev

%error

Vol %

Blended whiskey

16.5

33.6

39.1

0.21

-2.3%

40.0

Red wine

4.7

11.2

13.8

0.06

+2.2%

13.5

White wine

4.4

10.6

13.1

0.21

+0.8%

13.0

The final quantitative experiment that was done was determination of the acetone/water content of several different mixtures sold as fingernail polish remover (9). Each contained components other than acetone and water, but since these were not observed in the NMR specrtra, we assumed acetone and water added to 100%. The acetone signal is given in Figure 1 and the water OH signal is well separated. Taking into account the 6:2 ratio of protons in acetone compared to water, converting integrals to mol%, then mass percent and volume % was straightforward. Two brands, A and B, each with different sub designations were used. The results are given in Table 4. Clearly, Brand A has more acetone, the “active ingredient” in fingernail polish remover. 99 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Table 4. NMR Analyses of Fingernail Polish Remover


Mole %

Mass %

Volume %

acetone

water

acetone

water

acetone

water

Brand A regular

48

52

61

39

66

34

Brand A strengthening

46

54

59

41

64

36

Brand B regular

36

64

48

52

54

46

Brand B strengthening

35

65

48

52

54

46

Brand B nourishing

36

64

48

52

54

46

Another fingernail polish remover was listed as a “non-acetone” polish remover. A number of components were listed on the label, among them ethanol, ethyl acetate and water. We had obtained the spectra of ethyl acetate and ethanol as model compounds for chemical shifts and spin-spin splitting (Figures 3 and 4). The NMR spectrum in Figure 8 allowed identification of these two components. Fortunately, the acetate methyl group yielded an isolated singlet and the ethanol CH2 quartet was not overlapped. Taking into account the 3:2 ratio of the protons, it was possible to determine the relative number of moles of these two components. The area of the OH peak was then corrected for the contribution due to ethanol and the amount of water determined. Then it was possible to calculate mole %, mass % and volume % from the molecular masses and densities. The results are given in Table 5.

Figure 8. NMR spectrum of “non-acetone” fingernail polish remover. 100 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Table 5. NMR Analysis of Non-Acetone Fingernail Polish Remover Mole %

Mass %

Volume %

water

60

28

25

ethyl acetate

20

47

47

ethanol

20

25

28


Structure: Identification of Unknowns In addition to the types of quantitative applications above, the freshman groups were also introduced to simple structure determinations. The relationships between chemical shifts, multiplicity and structure were discussed in detail for the six model compounds: the two ketones, two alcohols and two esters.

Figure 9. Chemical shifts (ppm) for protons in model compounds.

Figure 10. Predicted multiplicity for model compounds. Figures 9 and 10 were constructed in the lecture room by having the students review the spectra in Figures 1, 3 and 4. Each group of students was given six unknown samples to work on as a team. They were also given the structures of the eight molecules in Figure 11 and told that the six samples were included in this list. The students were told to prepare a chart of possible ranges of chemical shifts and multiplicities for the protons in this list of compounds based on comparison to the model compounds in Figures 9 and 10. For alcohol samples, they were to include splitting with and without exchange. There was a discussion of equivalent protons and stepwise application of the 2nI+1 rule for nonequivalent protons. Each student ran two samples. There was some duplication. 101 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 11. Eight possible structures for the six unknown samples.

Figure 12. NMR spectra of dry isopropyl alcohol (top) and isopropyl alcohol after addition of dilute HCl.

They were to add a small amount of HCl if they suspected an alcohol. The students compared results, they made Power Point presentations and explained the reasoning behind their choices. Most of the assignments were quite straightforward. Some required a little more thought. Figure 12 contains spectra of isopropyl alcohol, with and without the addition of a drop of dilute HCl. The collapse of the splitting with the addition of acid, greatly simplified the interpretation. Figure 13 is the one example that proved most challenging because the difference in chemical shifts of two methyl triplets with essentially the same coupling constants caused them to overlap and appear to be a quartet. This example led to the most spirited discussions among the students. 102 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 13. NMR spectrum of ethyl propionate with overlapping triplets giving an apparent quartet.

More Advanced Applications Time Averaging and Exchange Reactions Time averaging and proton exchange were discussed earlier with respect to ethanol (Figure 4). The kinetics of base-catalyzed deuterium exchange of the alpha protons of pentanones and hexanones were studied. This served as a complement to a GC/MS experiment on ketones that has been part of the organic chemistry laboratory course for a number of years. Three different cases arose: 1) those where the NMR signals were so badly overlapped and the disappearance of protons could not be followed, e.g. 2-methyl-3-pentanone, 2) those where the disappearance of methyl or methylene protons could be followed independently, e.g. 2-pentanone, 3) those where a methine proton was either too weak or too split to be reliably followed, e.g. 3-methyl-2-butanone in which case a different strategy was employed. In case 2) above, the derived rate constants for all the methyl and methylene exchanges for all the molecules were found to be approximately the same within a factor of 1.5. For case 3, the difference between the exchange of the methyl protons and the methine proton approached a factor of 10. This was more interesting and is described in detail below for 3-methyl-2-butanone. The spectrum and the exchange reactions are given in Figure 14. The experiment was performed by adding 30 microliters of ketone to 0.7 mL of D2O (99.9%D) in an NMR tube, then 10 microliters of 40% KOD in D2O(98+%D) and quickly mixing. Under these conditions, it was possible to follow the pseudo-first order decay of the methyl protons in a straightforward fashion. The disappearance of the methyl group is shown in Figure 15. The concentration is normalized to the concentration at time t=0. 103

Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 14. Exchange reaction and NMR spectrum of 3-methyl-2-butanone.

The rate of exchange of the methine proton was considerably slower than that of the 1-methyl protons. The methine proton resonance was 1/3 the intensity of the 1-methyl protons making it difficult to follow as the exchange proceeded. The methine proton was split into a septet, making the disappearance even more difficult to follow. However, there was another way to follow the disappearance of the methine proton. The methine proton splits the isopropyl methyls into a doublet with a coupling constant of ~ 7Hz. As the proton is replaced by a deuteron, the doublet disappears and a triplet (i.e. 2nI+1=3 when n=1 and I=1) with a splitting of ~ 1 Hz appears (Figure 16). It was possible to measure both the disappearance of the doublet and appearance of the triplet. Figures 15 and 17 clearly show almost a factor of 10 difference in the rate of exchange of the three 1-methyl protons and the one methine. This led to discussion of the effects of steric hindrance and the relative stability of the different enolate intermediates involved in the exchange.

Figure 15. Disappearance of the [CH3] signal with time for 3-methyl-2-butanone. 104 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Figure 16. Disappearance of the doublet (CH3)2CH and appearance of the triplet (CH3)2CD for 3-methyl-2-butanone.

Figure 17. Time dependence of the doublet, (CH3)2CH, and the triplet, (CH3)2CD, for 3-methyl-2-butanone. Time Averaging: NMR vs Vibrational (Raman) Time Scales The next experiment has been performed in the physical chemistry laboratory course, although it could easily be adapted into instrumental analysis course. It was a challenge for two to four students, working as a team, to complete it in one 4-hour lab period. It allowed the illustration of different time scales between NMR and vibrational spectroscopy. Phosphates are encountered in many areas of chemistry. Phosphoric acid is one of the first examples of a polyprotic acid encountered in General Chemistry courses. The use of phosphate buffers is important in biology and biochemistry as well as in separation science, e.g. High Performance Liquid Chromatography (HPLC) (10). The importance of organic phosphates in biochemical systems cannot be overemphasized. The interaction of various metal ions with phosphate species has been studied by 31P NMR (11). The transformation of ATP/ADP/AMP and inorganic phosphate has been studied by 31P NMR in-vivo in muscle, livers and heart research (12). Raman spectroscopy of biological systems has been applied almost since the first application of lasers (13). Although FTIR 105



has been applied to the study of aqueous phosphates, the high absorbance of water compared to the low scattering cross section of water has made Raman spectroscopy a preferable technique for many biological systems. This experiment was developed to introduce undergraduate students to simple applications of 31P NMR and Raman spectroscopy to the transition among various aqueous orthophosphate species as a function of pH. These experiments were performed with a pH probe, an Anasazi Eft 60MHz FTNMR and a Delta Nu Advantage 633 Raman spectrometer, all of which might be accessible to smaller departments such as at UST. As a result of the limitations of instruments available, we were limited to using higher concentrations of phosphate solutions. Potassium phosphate was chosen because it has the highest solubility over the entire pH range. Although saturated potassium phosphate at high pH is in excess of 3M, it drops to a bit over 1M at pH between 5 and 7. An aqueous solution of hydrated tripotassium phosphate, ~ 1M was made, and its pH measured. The concentration was determined by titration with a standardized HCl solution. A phosphoric acid solution of the same concentration was used to lower the pH of the phosphate solution in increments of ~0.5 pH units which maintained a constant total phosphate concentration. A sample, large enough for NMR, was removed at each 0.5 pH value and the 31P chemical shift measured against an 85% phosphoric acid solution (chemical shift = 0.00) as a chemical shift standard. The Raman spectrum of the same solution in the NMR tube was also obtained. A plot of the chemical shifts vs pH and the individual Raman spectra were compared to the concentrations calculated from the known pKa values and to Raman spectra of phosphate species reported in the literature (14).

Figure 18. pKa values for phosphoric acid (top) and concentrations as a function of pH for a 1.2M solution. 106 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


Because very small chemical shift differences are involved, care had to be exercised to obtain data of sufficient precision. The temperature in the magnet in the Eft 60 is maintained at 30°C. The samples in the NMR tubes were equilibrated in a water bath maintained at that temperature just prior to measurement. Since the Eft 60 has no lock, a standard of 85% phosphoric acid was run after each 2-3 samples and the zero point reset if necessary. After the phosphate solution was titrated with HCl and diluted to 1.2 M, the pKa values in Figure 18 were used to calculate the concentrations of the various phosphate species as a function of pH, also shown in the Figure. Figure 19 shows the 31P signals at several of the pH values. A broadening of the NMR signal is observed approaching pH 7, but in no case is more than one signal present. The conclusion is that the average chemical shift is observed for two species that interconvert rapidly compared to the difference of the chemical shifts of the two species.

Figure 19.

31P

spectra of phosphate solutions at selected pH values.

Figure 20 shows the variation of the chemical shift with pH, measured at 0.5 pH unit increments, and also shows the titration curve for the phosphate solution with HCl. It is seen that the flat regions of the curve in Figure 18, near pH 10 and 5, correspond to steep regions in Figure 20, where single species are present over a range of pH values. Where the slope of the curve in Figure 20 is flatter, a small change in pH corresponds to a large change in chemical shift. In this range Figure 18 shows the presence of two species and a significant change in concentration from one species to the other over a very small pH range. From the calculated concentration curve, it is seen that the rapid changes in concentration occur at pH values of about 12-12.5, 6.5-7 and 2, corresponding approximately to the pKa values. On the other hand, at a pH near 10 and also near 5, the change in pH has little effect on the concentration of the species. These correspond to the inflection points in the two curves in Figure 20 and represent the flat parts of the concentation curves in Figure 18. The conclusion reached is that the chemical shift observed for the single line in a phosphate solution represents an average of the concentration-weighted species present. Measurement of the chemical shift allows estimation, albeit not the best or easiest way, of the pH of a phosphate solution. Parallel to the NMR study, Raman spectra were obtained from the same samples in the NMR tubes at intervals of 0.5 pH units. Figure 21 are the Raman spectra at pH values where there is essentially one species present. The spectra, which arise from primarily P-O and P-O-H vibrations (14), are markedly different. 107


31P

Chemical shift variation with pH (left) and titration curve (HCl) (right) for phosphate solutions.


Figure 20.

Figure 21. Raman spectra of individual phosphate species at the indicated pH values. Frequencies are in cm-1. A drop of concentrated HCl was added at the end to lower the pH to 0.2. The transition between species can be seen by examining the spectra of solutions corresponding to regions where two species are present at the same time (Figure 22) compared to the spectra of individual species in Figure 21. The spectrum at pH 12 is a mixture of those at pHs 13.4 and 10; pH 6.5 is a mixture of those at 10 and 5; pH 2 is a mixture of pH 5 and 0.2. This experiment demonstrated several things to students. It requires careful pH measurements. Although sensitivity can be an issue with a 3mW helium neon laser Raman instrument and sensitivity and precision with a 60 MHz permanent 108 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


magnet NMR with no lock, careful attention to detail yields reasonable data. The experiment clearly illustrates the different time scales between NMR, which sees an average of species vs Raman which observes individual species (14). In addition to its use as a physical chemistry experiment, this has also been used as a special project for freshman students during or after their second semester.

Figure 22. Raman Spectra of solutions at pH values where two species are present. Frequencies are given in cm-1. 31P

NMR of Biologically Important Molecules and NMR Simulation

We had extended the study of phosphate molecules into a set of undergraduate research projects. We continued our study of phosphates for several reasons. While our low field was a disadvantage, it allowed (required) us to address examples of higher order spectra and to introduce NMR simulation. We also pointed out that spectra obtained at higher fields would not be as complicated, although we later came across examples where low-field experiments are actually less complicated. Proton decoupling was also introduced. As seen above, the NMR spectrum of phosphate is a single line between 0 and just > 5ppm depending on the pH. The position depends on the relative amounts of the species present. Pyrophosphate (P2O74-) has 2 equivalent phosphorous atoms and also gives a single line, but with a negative chemical shift relative to 85% phosphoric acid. Triphosphate has two equivalent phosphorous atoms with a unique phosphorous between them. The first order spectrum expected for the triphosphate anion would be a doublet for the two equivalent phosphorus atoms and a triplet for 109



the one in the center. That is approximately what is observed, but with additional splitting due to second order effects. This was reproduced by simulation with the gnmr program (15, 16). In addition we have studied adenosine mono- di- and tri- phosphates (AMP, ADP and ATP). The conversion of ATP to ADP releases significant energy. Among other things, this provides the driving force in reactions involving metabolism. ATP, ADP and AMP are interconverted. The spectrum of AMP is a single line, with a pH dependent chemical shift. The non-equivalent P atoms in ADP yield a second order spectrum, a classic AB pattern with Δδ (in Hz) = 16.5Hz, almost equal to 2JPP= 19.7Hz shown in Figure 23. With the J/Δδ = 1.2, the two chemical shifts are clearly not at the midpoint of each doublet. This was a good example of a molecule with a second order spectrum at 60 MHz that would be essentially first order at 500 MHz. For ATP, the appearance of two doublets and a triplet is misleading (Figure 24). The coupling of the central β P is essentially identical to the α (18.2 Hz) and γ (18.4 Hz) P atoms. Consequently, the components of the two doublets of the β P overlap. As the pH is lowered, the chemical shifts all move to higher field and the lines broaden. The γ P is the most affected and at a pH of ~ 5-6, the α and γ signals merge. At very low pH, ~ 2, the splitting can no longer be seen in any of the signals.

Figure 23.

31P

spectrum of ADP. The smooth line is the least squares fit superimposed over the experimental spectrum.

Figure 24.

31P

spectrum of ATP at high pH. 110



The last 31P example that we examined was phosphoenolpyruvate (Figure 25). This was a case which was baffling at first. It was also the first case where we had heteronuclear coupling and longer range than two bond coupling. However, even with this, we expected the interpretation to be straightforward. In D2O solution, we expected two similar, but different protons that would couple to each other 2JHH, and two different 4JPH1, 4JPH2 couplings. Based on these assumptions, we predicted the protons would couple and produce two doublets at different chemical shifts and that each doublet would be split again, by the phosphorous, but with different splittings. What was obtained appeared to be two triplets. The 31P spectrum, without proton decoupling was expected to be a pair of doublets, but what was observed appeared to be a triplet as seen in Figure 25. A key, however, was that the proton decoupling experiment yielded a single line in the 31P experiment.

Figure 25. Proton spectrum (top), and 31P spectra without (left) and with (right) proton decoupling for phosphoenol pyruvate. Simulated spectra are shown with a linewidth of 0.001 Hz. This is what led us to decide that 4JPH1 ~ 4JPH2 = 2.1 Hz. What appeared to be two triplets were pairs of doublets, overlapping in the center which accounted for the appearance in the 31P spectrum. The next conclusion was that the 2JHH were nearly equal to the 4JPH. We used the gnmr program (16) to simulate the spectra and derived the following parameters: 2JHH = 2.3Hz and the proton chemical shifts are 5.831 and 5.484 ppm with the phosphorous chemical shift -4.587 ppm. With these parameters, the simulated spectra were essentially perfect fits to the experimental. The simulated spectra are shown in Figure 25 with a linewidth of 0.001 Hz to show the two lines in the center that add to give the appearance of a triplet. 111 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


2D NMR Experiments A laboratory experiment was developed to introduce students in either an organic chemistry or biochemistry lab course to two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy using simple biomolecules (17). The goal of this experiment is for students to understand and interpret the information provided by a 2D NMR spectrum. Biological molecules were chosen for an advanced NMR exercise that involves 2D NMR analysis with a low-field NMR instrument (60 MHz). Details are given in Reference (17). Each experiment involved the students analyzing three samples: two single pure amino acids and a dipeptide made up of the two different amino acids. The samples were all run in D2O solutions acidified with DCl in order to fully dissolve the sample (with the exception of samples containing tyrosine where NaOH was needed for solubility). No clearly resolved amine, amide, carboxylic acid, alcohol, or sulfhydryl functional group protons are observed because of replacement of protons with deuterium. This simplified the spectra but also limited the amino acids that work best in this lab experiment; amino acid side chains with these functional groups were generally avoided. Amino acid combinations of methionine, valine, isoleucine, phenylalanine, alanine, glycine, proline, tyrosine, and leucine worked best and were routinely used in this lab experiment.

Figure 26. 1H NMR spectra of the Phe-Ala (top) and Ala-Phe (bottom) dipeptides. Reproduced with permission from Reference (17). Copyright 2015, ACS. The students identified their two amino acids from the 1H experiment, then obtained a 1D 1H spectrum of their dipeptide. Examples of the student generated spectra of the two phenylalanine and alanine dipeptides are shown in Figure 26. All of the proton peaks are assigned; αHA refers to the α-proton on the alanine amino acids, and αHF refers to the α-proton on the phenylalanine amino acids. The NMR spectra are complex and have apparently overlapping peaks, and further analysis is provided by the COSY spectrum. The COSY experiment 112 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


required approximately 20 min in the 60 MHz instrument. The COSY spectrum for each dipeptide (Figure 27) was used to assign the α-proton peaks.

Figure 27. COSY spectra of Phe-Ala (left) and Ala-Phe (right). Reproduced with permission from Reference (17). Copyright 2015, ACS. The information obtained from the COSY spectrum was in the form of the off-diagonal cross peaks that served as evidence that these protons were 2 or 3 bonds apart. These off-diagonal peaks are the result of spin coupling between groups of protons. In each panel, the off-diagonal peaks for the α-protons are labeled with dotted lines that show the α-H on the Phe(F) amino acid that gives an off-diagonal peak through its interaction with the CH2 group on the F side chain. The dashed line shows the α-H on the Ala(A) amino acid that gives an off-diagonal peak through its interaction with the CH3 group on the A side chain. In Ala-Phe, the α-proton at ~3.7 ppm was the alanine α-proton, and the proton at ~4.2 ppm was the phenylalanine α-proton (Figure 26). In Phe-Ala, the signals for these two protons overlap; this is particularly apparent in the COSY spectrum. HETCOR analysis was done as an independent research project. First it was necessary to obtain the 13C spectra of the dipeptides (Figure 28). Each of these was an overnight run. Since the Eft 60 has no lock and is subject to frequency drift, Block Averaging with Peak Registration (BAPR) and a higher concentration was used. Each spectrum in Figure 28 was the average of 100 blocks of 100 scans.

Figure 28.

13C

spectra of Phe and Ala-containing dipeptides. Reproduced with permission from Reference (17). Copyright 2015, ACS. 113



The HETCOR protocol (Figure 29) was run with a relaxation delay of 2 seconds and 32 scans. For Ala-Phe, note that the pair of 1H and 13C α-CH chemical shifts for Ala noticeably differ from the pair of 1H and 13C α-CH chemical shifts for Phe. In contrast, for Phe-Ala, these two contours noticeably differ only in their 13C chemical shifts, as each α-CH gives a 1H signal at 4.2 ppm.

Figure 29. HETCOR plots for Phe-Ala (left) and Ala-Phe (right) dipeptides. 1H spectra are plotted vertically and 13C horizontally. Reproduced with permission from Reference (17). Copyright 2015, ACS. The HETCOR analysis is not routinely included in the lab experiment for the biochemistry lab course because of the length of time required and the larger class size. These results demonstrate that meaningful 2D experiments may be performed with 60 MHz instruments if the systems are carefully chosen.

Simple Organofluorine Molecules with Complex Spectra Analyzed by Spectral Simulation Access to a variety of organofluorine compounds from a synthesis project gave us the opportunity to use the multinuclear capability of the Eft 60 for a series of related molecules. This started with an undergraduate research project for one student and led to involvement of other undergraduate students over several years, collaboration with personnel at Rice and the use of high field instruments through the Shared Equipment Authority at Rice. Additional molecules were obtained from Synquest Laboratories (www.synquestlabs.com) In the initial project 1H, 13C (proton decoupled) and 19F spectra of three triflates, 2,2,2-trifluoroethyl, 2,2,3,3,3-pentafluoropropyl and 1,1,1,3,3,3-hexafluoroisopropyl triflate, were obtained (Figure 30). The samples were run neat with a small amount of TMS. The chemical shifts and coupling constants derived from the 60 MHz data are shown in Table 6. There was one exception, those underlined were determined from high field data from the instruments at Rice. 114



Figure 30. (I)2,2,2-trifluoroethyl-, (II)2,2,3,3,3-pentafluoropropyl- and (III)1,1,1,3,3,3-hexafluoroisopropyl- triflates. The first two molecules provided interesting and informative exercises in interpreting first order spectra (18). The first, 2,2,2-trifluoroethyl triflate, was fairly straightforward. The coupling between the fluorines on carbon 2 and the protons was observed in both 1H and 19F spectra. Fluorine is known to exhibit long range coupling, which makes these molecules more interesting and a small 6-bond coupling (~0.9 Hz) between the fluorines on carbon 2 and triflate methyl fluorines was observed.

Table 6. Chemical Shifts and Coupling Constants Determined for (I) 2,2,2-Trifluoroethyl, (II) 2,2,3,3,3-Pentafluoropropyl, and (III) 1,1,1,3,3,3-Hexafluoroisopropyl Triflates (Figure 30) I δC1= 70.38 ppm δC2= 123.02 ppm δCtr= 120.55 ppm δF2= -75.76 ppm δFtr= -75.26 ppm δH= 4.71 ppm

II

III

δC1= 67.25 ppm

2JC1F2=

29.2 Hz

δC1,3= 121.35ppm

δC2= 110.24 ppm

1JC2F2=

-257.1 Hz

δC2= 76.85 ppm

δC3= 117.70 ppm

2JC2F3=

39.1 Hz

δCtr= 120.53 ppm

δCtr= 118.23 ppm

1JC3F3=

-285.1 Hz

δF1,3= -75.32ppm

δF2= -75.77 ppm

2JC3F2=

33.9 Hz

δFtr= -75.57ppm

δF3= -125.28 ppm

1JCtrFtr=

2JC1F2=

39.9 Hz

δFtr= -85.14 ppm

3JHF2

1JC2F2=

-276.4 Hz

δH= 4.82 ppm

3JF2F3

1JCtrFtr= 3JHF2

-318.1 Hz

= 7.5 Hz

6JF2Ftr

-318.0 Hz

= 11.8 Hz = ~1.2 Hz

δH= 5.36 ppm 1J C1,3F1,3= -282.0 Hz 2JC2F1,3=

36.9 Hz

1JFtrCtr=

-318.0 Hz

3JHF1,3

= 5.2 Hz

6JF1,3Ftr

= ~0.9 Hz

= 2.6 Hz

The 1H and 19F spectra from 2,2,3,3,3-pentafluoropropyl triflate were straightforward. The proton decoupled 13C spectrum was more complex. Carbon 1 was the easiest, since it was a triplet with 2-bond coupling (29.2 Hz) and separated from the rest. The triflate carbon gave a quartet (-318.0 Hz) as expected from 1-bond coupling with the three fluorines. The others were more complicated. Carbon 2 was a triplet (-257.1 Hz) from 1-bond coupling to CF2 fluorines and each component was a quartet (39.1 Hz) from 2-bond coupling to CF3 fluorines 115 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


on carbon 3. Carbon 3 was a quartet (1-bond = -285.1 Hz) of triplets (2-bond = 33.9 Hz). Unraveling the various patterns of lines in the spectrum was a good teaching opportunity. The last compound in this group, 1,1,1.3,3,3-hexafluoroisopropyl triflate (III in Table 6) originally appeared to be quite straightforward. The proton spectrum gave a septet as expected from the six fluorines. This is shown in Figure 31; 3JHF of 5.2 Hz was easily derived.

Figure 31. 1H spectrum of 1,1,1,3,3,3-hexafluoroisopropyl triflate. The proton decoupled 13C spectrum of III (Figure 32) was simpler than that of II. The feature that was different was the underlying structure to the quartet from carbons 1 and 3 which arose because the two trifluoromethyl groups are no longer equivalent and a 3JCF coupling was possible with the 12CF3 group.

Figure 32. Proton decoupled 13C spectrum of 1,1,1,3,3,3-hexafluoroisopropyl triflate. The 19F spectrum, however was a real puzzle on the 60 MHz instrument. We expected a doublet with a splitting of 5.2 Hz for the isopropyl fluorines and a singlet for the triflate fluorines possibly split again by a 6-bond 6JFF since a 6-bond coupling was observed for compound I. However, a complex higher order pattern was observed with lines separated by ~2.6 Hz (Figure 33). The spectrum from the 500 MHz instrument at Rice yielded an interpretable first order spectrum (Figure 33). The triflate fluorines were a septet with a splitting of 2.6 Hz. The isopropyl fluorines appeared as six lines rather than as a doublet of quartets. This arose because 3JHF = 2 times (6JFF) and the two quartets (6JFF =2.6 Hz) were offset by the 116 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


5.2 Hz 3JHF . The resulting 1:3:4:4:3:1 intensity pattern was consistent with this interpretation. At 60MHz, the chemical shift difference, 0.25 ppm, translates to ~ 14 Hz, reasonably close to the coupling constants. At 500 MHz, this difference is ~ 118 Hz leading to a first order spectrum. The 500 MHz spectrum was obtained with a tube-in-a-tube with the triflate in the coaxial inner tube and acetone-d6 in the outer tube (for locking and shimming). The chemical shifts and coupling constants determined were used as input to the gnmr (16) program to simulate the spectra at both fields. The correspondence was excellent as shown in Figure 33.

Figure 33. Experimental and simulated 19F spectra of 1,1,1,3,3,3hexafluoroisopropyl triflate obtained at 60 MHz and 500 MHz. Once high field instruments were employed, several effects that were common to molecules with hexa- or heptafluoro- isopropyl groups were observed. The complex line shapes in the 13C spectra and the complex and different line shapes for the 13C satellites in the 19F spectra originate in the inequivalence of the hexafluoroisopropyl CF3 groups when one is 12CF3 and the other is 13CF3. These line shape effects depend in a complex manner on the size of 1JCF, 3JCF, and 4JFF; on the magnitude of the one-bond 13C/12C isotope effect on the 19F chemical shift; on the field strength; also on 6JFF in the case of the triflate and the chemical shift differences in Hz between the various 19F signals. Cross-correlated relaxation results in different 13C spin-lattice and spin-spin relaxation rates for various transitions of the hexafluoroisopropyl group. As a result in the 13C spectrum, the complex, higher order group of signals for each part of the hexafluoroisopropyl CF3 quartet exhibits peak height and linewidth variations within a group. Spectral simulation with the gnmr program (16) was essential to understanding the 117



complex line shapes in the 13C satellites in the 19F spectra and in the 13C spectra of the 13CF3 groups. Figure 34 gives the structures of organofluorine molecules for which we have completed detailed analyses including determination of chemical shifts and coupling constants for the common isotopomers, and analysis of the 13C spectra and 13C satellites in the 19F spectra of 13CF3 groups. To study cross-correlated relaxation, series of 13C T1 inversion-recovery spectra of dilute solutions of some in the list have also been obtained. ortho-CF3 substituted benzenes exhibit very similar behavior to hexa- and heptafluoro isopropyl compounds and two have been studied. The analyses of those molecules in the first row have been published (19, 20). The others have been presented at ACS meetings.

Figure 34. Organofluorine molecules for which detailed analyses of 13C spectra and 13C satellites of 13CF3 groups have been completed. Figure 35 illustrates the 13C isotope shift which brings the high field member of the 13CF3 signal close to the 12CF3 signal in the same molecule, which in turn leads to higher order effects, especially for the high frequency component.

Figure 35. Illustration of the 13C isotope shift for 19F leading to higher order interactions. 118 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


The impact will be illustrated for 1,1,1,3,3,3-hexafluoroisopropyl alcohol, rather than the triflate, because it is simpler. The necessity of use of a spectral simulation program such as gnmr (16) will be evident. After the shifts and coupling constants for the all-12C isotopomer had been determined, the substitution of 13CF3 was made in the simulation and the appropriate shifts and coupling constants included and adjusted. The relevant chemical shifts and coupling constants are given in Table 7. The experimental and simulated high and low frequency 13C satellites in the 19F spectrum are shown in Figure 36 (19). The half-width used in the simulation was 0.65 Hz. The simulation with a line-width of 0.01 Hz scaled to fit on the figure is shown to indicate overlapping lines. The agreement between the experimental and simulated 13C satellites in the 19F spectrum of this molecule is excellent, with root mean square (RMS) deviations of 0.14 and 0.18 Hz for the 18 and 8 welldefined signals in the high frequency and low frequency satellites.

Table 7. Chemical Shifts and Coupling Constants Used for the Simulations for 1,1,1,3,3,3-Hexafluoroisopropyl Alcohol 12CF3

- δF

-75.682 ppm

3JHF

6.55Hz

13CF3

- δF

-75.806 ppm

1JCF

-281.55Hz

13CF3

– δC

123.365 ppm

3JCF

2.40 Hz

4JFF

9.20 Hz

Figure 37 shows an expanded view of the four regions of the 13C spectrum. The experimental regions are shown above and the simulated are shown below. The half-width used in the simulation was 0.5 Hz. The simulation at 0.01Hz was also included. The agreement between the experimental and simulated CF3 signals in the 13C spectrum is excellent with respect to the frequencies of the various signals (with RMS deviations of 0.21, 0.12, 0.05, and 0.14 Hz for the 5, 10, 9, and 5 signals in the four groups of CF3 signals) but the height of some of the signals clearly differs in the experimental and simulated spectra. This is more extensively discussed in reference (19). The 13C T1 inversion-recovery experiment on a dilute solution indicated the occurrence of cross-correlated relaxation because the four groups of signals did not relax at the same rate. A plot of the most critical inversion-recovery spectra for different τ values is shown in Figure 38 (19). Cross-correlated relaxation was also observed for both the triflate 13CF3 and the hexafluoroisoprpyl 13CF3 in 1,1,1,3,3,3hexafluoroisopropyl triflate. Additional inversion-recovery experiments showed this same phenomenon for 13C satellites of triflic anhydride, both in the 13C spectra and 13C satellites in the 19F spectra of hexafluoroacetone and in the 13C spectra of perfluoro-t-butyl alcohol (20).



Figure 36. 13C high (a,c) and low (b,d) frequency satellites in the 470.5 MHz 19F spectrum of 1,1,1,3,3,3-hexafluoroisopropyl alcohol. Experimental (a,b) and Simulated (c,d). Reproduced with permission from Reference (19). Copyright 2010, Wiley.

Figure 37. 125.8 MHz 13C spectrum of 1,1,1,3,3,3-hexafluoroisopropyl alcohol. Experimental (a-d) and simulated (e-h). Reproduced with permission from Reference (19). Copyright 2010, Wiley.



Figure 38. Vertically expanded plots of the CF3 signals in a series of 13C T1 inversion-recovery spectra of a dilute solution of 1,1,1,3,3,3-hexafluoroisopropyl alcohol at the τ values near where the CF3 signals are nulled: (a) 8 s, (b) 9 s, (c) 10 s, (d) 12 s. 125.8 MHz 13C. Reproduced with permission from Reference (19). Copyright 2010, Wiley.

As an illustration of the utility of spectral simulation in assigning and analyzing complex splitting patterns, several examples of 13C satellites in 19F spectra and the complex splitting of 13CF3 quartets in the proton decoupled 13C spectra are given below. These include 1,1,1,3,3,3-hexafluoroisopropyl triflate (Figures 39,40), hexafluoroacetone (Figures 41,42) and perfluoro t-butyl alcohol (Figures 43,44).



Figure 39. Comparison of the experimental(a,b) and simulated (c,d) high and low frequency hexafluoroisopropyl 13C satellites in the 470.5 MHz 19F spectrum for 1,1,1,3,3,3-hexafluoroisopropyl triflate. Reproduced with permission from Reference (19). Copyright 2010, Wiley.

Figure 40. Comparison of the experimental(a-d) and simulated (e-h) 125.8 MHz 13C spectrum for 1,1,1,3,3,3-hexafluoroisopropyl triflate. Reproduced with permission from Reference (19). Copyright 2010, Wiley.



Figure 41. Comparison of the experimental (a,b) and simulated (c,d) high and low frequency 13C satellites in the 470.5MHz 19F spectrum for hexafluoroacetone. Reproduced with permission from Reference (20). Copyright 2012, Elsevier.

Figure 42. Comparison of the experimental (a-d) and simulated (e-h) 125.8MHz spectrum for hexafluoroacetone. Reproduced with permission from Reference (20). Copyright 2012, Elsevier.

13C



Figure 43. Comparison of the experimental (a,b) and simulated (c,d) high and low frequency 13C satellites in the 470.5MHz 19F spectrum for perfluoro-t-butyl alcohol. Reproduced with permission from Reference (20). Copyright 2012, Elsevier

Figure 44. Comparison of the experimental (a-d) and simulated (e-h) 125.8MHz 13C spectrum for perfluoro-t-butyl alcohol. Reproduced with permission from Reference (20). Copyright 2012, Elsevier

The comparison between experimental and simulated spectra is excellent giving a great degree of confidence in the coupling constants derived. These molecules, along with the others listed in Figure 34, have provided excellent opportunities for instruction in interpretation and analyses of complex spectra through the use of spectral simulation. 124 Soulsby et al.; NMR Spectroscopy in the Undergraduate Curriculum: Upper-Level Courses and Across the Curriculum Volume 3 ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

Conclusions


A 60 MHz Eft 60 FT NMR with multinuclear capability may be employed, with careful choices of systems, to give meaningful experience to undergraduate students both in laboratory course work and undergraduate research. The access to high field NMR and NMR expertise at a nearby major research university is invaluable. Through the academic rates for instrument time available through the Shared Equipment Authority at Rice University, the faculty and students at the University of St. Thomas have a valuable resource. At the same time, the participation of students and faculty from UST broadens the impact of the excellent facilities at Rice.

Acknowledgments The Anasazi Eft 60 was purchased through NSF CCLI Grant Award No. 0536648. The Welch Foundation is acknowledged for support at UST through Departmental Grant AV 0024. Funding for the 400- and 500- MHz spectrometers at Rice University was provided through NSF awards CHE-075728 (400 MHz NMR) and CHE-9708978 (500-MHz NMR) and for the upgrade of the 500 MHz NMR through CHE-0947054. Additional funding for the 500 MHz broadband observe probe with 19F capability was provided through an AFOSR award FA9550-06-1-0424 (Strategic Partnership for Research in Nanotechnology). We especially want to thank the many students over the years who have participated through laboratory courses and especially those involved in undergraduate research projects.

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NMR at the University of St. Thomas (TX): Cooperation and

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