Determination of pk a using NMR spectroscopy

Transcription

1 Determination of pk a using NMR spectroscopy Objectives: 1. ecome familiar with how to collect and analyze data with the NMR 2. Understand why peaks in the NMR spectrum shift as the ph is changed 3. Understand acid/base equilibria 4. Determine the pk a of a methyl-substituted pyridinium cation 5. uild a model for the ph dependence of a proton chemical shift ackground: Compounds in aqueous solution can exist in multiple forms. An example of this is the equilibrium between pyridine and the pyridinium cation as shown in equation 1. The ratio of these two forms is dependent on the ph of the solution. At low ph, it will exist almost entirely as H, and at high ph, it will exist almost entirely as. In previously performed laboratories at UNO (General Chemistry II and Physical Chemistry I) students have used both ph titrations and spectrophotometric methods to determine the K a of a compound. In this laboratory, the K a (or pk a ) will be determined using data collected from a nuclear magnetic resonance (NMR) spectrometer. N H H 2 O N H 3 O (1) H The dissociation constant for the pyridinium cation is given by equation 2. This equation can be manipulated, using logarithms, to obtain an expression relating the pk a of the pyridinium cation to the ph of the solution and the concentration ratio of the two species as shown by equation 3. K a [ H O ][ ] = 3 (2) [ H ] pk a [ H ] = ph log (3) [ ] NMR spectra will be used to obtain the concentration ratio of the two species. The NMR shift of the pyridinium ring hydrogens is dependent on the relative concentrations of H or. If the solution is at a low ph and the species is 100% protonated (H ), it will have a chemical shift of ν H. If the solution is at a high ph and the species is 100% deprotonted (), it will have a chemical shift of ν. If the solution is at a ph where both species are present, it will have a 1

2 chemical shift of ν. The chemical shift of ν is related to the chemical shift of the protonated and deprotonated forms by equation 4, ν = ν x ν x (4) H H where ν is the observed chemical shift at the specified ph, and x H and x are the mole fraction of the two species at the same specified ph. However, in order to determine the pk a of the pyridinium cation of interest, x H and x must be calculated using the chemical shifts of the compound. This is accomplished by using equations 5 and 6. Equation 5 uses the chemical shift of a specific ring proton to determine the mole fraction of the deprotonated species (x ) and equation 6 can be used to determine the mole fraction of the protonated species (x H ) since the sum of the two fractions must equal one. chemical shift at low ph - observed chemical shift x = (5) chemical shift at low ph -chemical shift at high ph xh x = 1 (6) After the mole fraction of the two forms has been determined, the pk a can be estimated using equation 7. Equation 7 is similar to equation 3 except that concentrations of the two species have been replaced by mole fractions. pk a x ph log H = (7) x Pre-lab activities: 1. What is the conjugate base to acid ratio for a solution of acetic acid adjusted to a ph of 4.00, 4.76, and How large (in megahertz) is the NMR spectrometer that is housed in the UNO chemistry department s instrument facility? What is the strength of the magnetic field of this NMR spectrometer? 3. What is a typical solvent used in preparing samples for NMR analysis? 4. FT-NMR spectrometers have a deuterium frequency-lock. What is a deuterium frequency-lock? Why is it important? 5. Draw the chemical structures for pyridine and 2,6-lutidine. In addition, find the pk a values for the conjugate acid forms of these two compounds and reference your source(s). Equipment: NMR, NMR tubes, NMR tube cleaning apparatus, ph meter, milligram balance, 16x100 test tubes, test tube rack, disposable pipets 2

3 Reagents: D 2 O, concentrated HCl, KOH pellets, tetramethylammonium iodide, and methyl-substituted pyridines (2,3-lutidine, 3,5-lutidine, 2,6-lutidine, 2-picoline). Procedure: 1. Preparation of sample solution: In a test tube, which will accommodate a ph meter probe, weigh approximately 75 milligrams of sample (assigned by your instructor) and 5-10 milligrams of tetramethylammonium iodide. Next, add ~5 ml of D 2 O to the test tube and mix well. 2. Preparation of HCl Solutions: Two HCl solutions will be prepared, ~1.0 M and ~0.1 M solutions, to adjust the ph of the sample solution. To prepare the ~1.0 M solution, add approximately 0.2 ml (six drops) of concentrated HCl (~12 N) to a properly labeled test tube. Then slowly add about 2.2 ml (66 drops) of D 2 O to the test tube and swirl to mix well. To prepare the ~0.1 M solution, add approximately 0.1 ml (three drops) of the previously prepared ~1.0 M HCl solution to a properly labeled test tube. Then add about 0.9 ml (27 drops) of D 2 O to the test tube and swirl to mix well. 3. Preparation of KOH Solutions: Two KOH solutions will be prepared, ~1.0 M and ~0.1 M solutions, to adjust the ph of the sample solution. To prepare the ~1.0 M solution, place ~ 112 mg of KOH into a properly labeled test tube. Then, add about 2 ml (60 drops) of D 2 O into this test tube. Mix well. To prepare the ~0.1 M solution, add approximately 0.1 ml (three drops) of the previously prepared ~1.0 M KOH solution to a properly labeled test tube. Then add about 0.9 ml (27 drops) of D 2 O to the test tube and swirl to mix well. 4. Adjusting ph of the sample solution: NMR spectra of the sample solution should be collected every 0.5 to 2.0 ph unit; every 0.5 ph unit when the chemical shift of the protons is rapidly changing a and every 1.0 to 2.0 ph units when the chemical shift is somewhat constant at high and low ph values. The ph of the sample solution can be adjusted by adding the prepared HCl or KOH solutions drop wise to the test tube containing the sample solution. After adding the HCl or KOH, determine the ph of the solution. If the solution is not at the desired ph, add more drops of HCl or KOH. Generally, the ~1.0 M solutions of HCl and KOH will be used most often when adjusting the ph of the sample solution. The ~0.1 M solutions of HCl and KOH may be used when adjusting the ph of the sample solution when it is already at a high (above 8.5) or low (below 4) value. 5. Acquiring NMR spectra: After the ph of the solution has been adjusted appropriately, the sample can be loaded into an NMR tube and a spectrum acquired using the NMR spectrometer. When this is complete, remove the liquid sample from the NMR tube and place it back in the test tube containing the sample solution. Then go back to step 4 to adjust to the next appropriate ph. One suggestion is to begin NMR data collection at a high ph, adjust the ph to a low value, and then adjust the ph back to a high value. 6. Determination of peak shifts at various ph values: After the spectra have been collected, process the data (Fourier transform the FID, adjust the phase, and set the ppm of the reference peak) to determine the peak positions for each set of equivalent ring protons at the various ph values. More details are described below in the Data Analysis section. a The ph range when the chemical shift will be rapidly changing is when the ph=pk a ± 1.0. Since you do not know the pk a of your sample, I am providing the pk a range for the various samples that will be analyzed during this laboratory. The samples will have a pk a in the range of

4 Data analysis: To process the data collected using the NMR, the NT-NMR program will be used. The free induction decay (FID) will be Fourier transformed and properly phased. In addition, the x-axis of the NMR spectrum will be calibrated by specifying the chemical shift of the tetramethylammonium protons. You can assume the protons of the tetramethylammonium ion have a chemical shift of ppm.(1) In order to see the tetramethylammonium proton peak and the sample proton peaks, you will most likely need to zoom-in on the baseline since the water proton peak will be significantly larger than the ring protons of the compound being studied. An example NMR spectrum of a pyridine is shown in Figure 1. After the x- axis of the NMR spectrum has been correctly calibrated b, the chemical shift for a proton peak can be determined and recorded in your lab notebook and in your electronic spreadsheet. Determine the shift for each set of equivalent ring protons in the spectrum at each ph. Note that each set of equivalent ring protons may have a splitting pattern. Choose one peak in the splitting pattern and consistently record the position of that peak at each of the different ph values. Figure 1. NMR spectra of pyridine at ph=6.53. (A) Full scale spectrum showing large water peak at 4.85 ppm. () Expanded scale spectrum showing tetramethyl ammonium reference protons at ppm and the aromatic ring protons between 7 and 9 ppm. Plot the chemical shift for each set of equivalent ring protons in your sample as a function of ph. An example graph is shown in Figure 2. Next, estimate the chemical shift of the ring proton when at a high ph and a low ph and record these values in your spreadsheet (cells E1 and E2 in Figure 3). For each data point, calculate the mole fraction of base (x ), the mole fraction of acid (x H ), and the pk a for that data point using the equations given in the ackground section of the laboratory. Use a spreadsheet for these calculations as shown in Figure 3. These calculated pk a values can be used to determine the pk a of your sample. Only use the calculated pk a from the rapidly changing portion of the curve to determine the pk a of the sample. c Furthermore, the pk a determined for each set of equivalent ring protons should give the same pk a value. Use the above information to determine the best pk a of your sample and be sure to include an estimate of uncertainty when you report your value. b Sometimes it may be difficult to determine which peak is the tetramethylammonium proton peak in order to calibrate the x-axis. If the correct peak is chosen, the large water peak should have an approximate shift of 4.8 to 4.9 ppm. c Note that if data points are used at the extreme ph values (not the rapidly changing portion of the curve) the uncertainty of the calculated pk a value increases significantly. 4

5 Next, construct a spreadsheet which models your experimental data using the equations given in the background section of the laboratory. This model will be able to predict the chemical shift of a ring proton as a function of ph given the chemical shift in acid, the chemical shift in base, and the pk a of the compound. An example spreadsheet is shown in Figure 4. Plot the predicted chemical shift as a function of ph on the same graph as the collected NMR data (Figure 2) that was previously plotted. Figure 2. An example graph of the peak position of a pyridine ring proton as a function of ph. The graph on the right includes the model prediction in addition to the NMR data. Figure 3. An example spreadsheet used to calculate x, x H, and pk a. 5

6 Figure 4. An example spreadsheet used to create the chemical shift model. This model should be able to predict the chemical shift of a proton given the chemical shift in acid, the chemical shift in base, and the pk a of the compound. Questions: 1. Find a literature value for the pk a of your sample (include citation) that was used in this experiment. How does this compare to your experimental results? Calculate the percent error. 2. Why did the chemical shift of the protons change as the ph changed and why were there not two separate peaks (for protonated and deprotonated forms) with the intensity of those peaks changing as the ph changed? 3. When calculating the pk a, why is it important to choose data points at ph values when the chemical shift is rapidly changing and not choose data points at large and small ph values? 4. Tetramethylsilane (TMS) is commonly used for a reference standard. In this experiment, tetramethylammonium iodide was used instead. Explain why TMS was not used. 5. How does the model fit the data that was collected by the NMR? Does the model do a good job of fitting the data? Explain any areas where the model does not do a good job of fitting the data. 6. Using the model, change the value of the pk a for your compound and see how it affects the results of your model. What range of pk a values will give a curve that fits the NMR 6

7 data reasonably well? How does this range correspond to the uncertainly in the pk a value you calculated? Cleanup: All solutions should be collected in a bottle labeled for disposal. Prepare a disposal tag for this bottle. NMR tubes need to be cleaned using the NMR tube washer. Ask your professor for instructions on its use. References: 1. National Institute of Advanced Industrial Science and Technology (AIST); Spectral Database for Organic Compounds (SDS). 7