NMR spectroscopy, as it is typically discussed, or at least how it is initially taught, tends to refer to static systems. Chemical shifts, integrations and multiplicities are typically reported on fixed structures. Take pyridine, for example, it will have a characteristic 1H NMR spectrum in D2O with: δ 8.53 (d, 2H, o-ArH), 7.84 (t, 1H, p-ArH), 7.53 (m, 2H, m-ArH) ppm.
There are external factors (e.g., concentration, temperature, pH), however, that can result in physical or chemical changes within the sample. These changes will manifest in the position of the chemical shift and are therefore easily observable in routine 1H NMR! For the case of pyridine, we will explore the effect of pH on the chemical shift.
Pyridine, of course, is a nitrogen base, so at low pH we expect that it will be protonated to yield pyridinium – its conjugate acid form. So in very acidic samples (such as pH = 1) the pyridinium form will be the sole species. In this case, we would expect a 1H NMR spectrum with resonances at: δ 8.87 (d, 2H, o-ArH), 8.44 (m, 1H, p-ArH), 7.98 (m, 2H, m-ArH) ppm. The differences in the pyridine/pyridinium conjugate acid/base pair spectra are depicted below:
In the intermediate pH range, on the other hand, we can expect that both pyridine and pyridinium will exist in the sample and there will be an equilibrium. This can be described by an acid dissociation constant (Ka) expression and rearranged to afford the Henderson-Hasselbalch equation.
So…..pyridine and pyridinium have unique chemical shifts can you expect to see both of them in an 1H NMR spectrum? No! Nothing complicated (or convoluted) like that! These species are in a fast equilibrium, so on the NMR time scale what you “see” is actually a weighted, time-averaged chemical shift. This means that the observed chemical shift is a function that depends on: (i) the amount of each species; and (ii) the chemical shift of each species.
Still sounds complicated? Well, okay, maybe a little bit, but this means that NMR provides an easy means with which to experimentally determine the pKa of a given equilibrium! By varying the solution pH, and noting the chemical shift, one can construct a titration curve – the equivalence point of which the molar fraction will be one and pKa will equal pH.
How??
Prepare a 5mL 1.0M HCl and KOH stock solutions in D2O, as well as a 0.1M KOH solution to be used to adjust sample pH. Once the stock solutions have been prepared, set them aside and concoct the sample by combing: 3mL of a 0.25M N(Me)4I D2O solution, 12mL of D2O, 2mL of 1.0M HCl in D2O and 0.25mL of pyridine (enough to make at least 20 samples). Ensure that this is mixed well and use a pH meter to determine the initial pH of the sample. If it is greater than one add the HCl stock solution until the desired pH is reached. Once at pH = 1, transfer ~0.7 mL of this sample into an NMR tube and label it with the appropriate pH.
Slowly raise the pH of sample solution using the KOH stock solution – transferring a 0.7 mL aliquot to an NMR tube at each pH step. Again – be sure to mix well in each measurement and record the pH at each step. At low and high pH (< 4 and > 7.5) these aliquots can be taken at 0.5 and 1 pH increments, however in the intermediate pH range, care should be prepare NMR samples at more regular intervals.
One the samples are prepared, 1H NMR spectrum can be taken with a set number of parameters (we chose a spectral width of 12 ppm, 0.5 s delay, and 32 scans).
Now what??
Load measured 1H NMR spectra into MNova for additional processing. Each spectrum should be phased, baseline corrected and referenced (to the tetramethylammonium idodide peak at 3.207 ppm). The chemical shift of pyridine is determined by selecting one of the peaks in a multiplet (either the ortho-doublet,meta-multiplet or para-triplet). It doesn’t matter which peak is selected (except that it must be resolved at each pH and is used consistently throughout the data analysis) and, in fact, can be done for all three resonances. The data shown below is for the downfield peak of the ortho-doublet. Once determined, this chemical shift was plotted versus the sample pH to afford a titration curve.