Determination of pKa using NMR spectroscopy
Adapted from Alan D. Gift,* Sarah M. Stewart, and Patrick Kwete Bokashanga, J. Chem. Educ. 2012, 89, 1458-1460
Background:
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 BH+, and at high pH, it will exist almost entirely as B. In this laboratory, the Ka (or pKa) will
be determined using data collected from a nuclear magnetic resonance (NMR) spectrometer.
+ + H2O + H3O+
N
N (1)
H
BH+ B
The dissociation constant for the pyridinium cation is given by equation 2. This equation
leads to the Henderson Hasselback equation relating the pKa of the cation (pyridinium cation) to
the pH of the solution and the concentration ratio of the conjugate pair. equation 3.
[ H 3O ][ B ]
Ka (2)
[ BH ]
[ B]
pH pK a log (3)
[ HB ]
NMR spectra will be used to obtain the concentration ratio of the two species (which is
the same as mole ratio for the conjugate pair in the same solution). The chemical shift of the
pyridinium ring hydrogens is dependent on the relative concentrations of BH+ or B. For a
solution at a low pH and the species in solution is ~100% protonated (BH+), and the chemical
shift are that of the protonated species, BH+. For a solution at a high pH the species in solution
is ~100% deprotonated (B), and the chemical shifts arte that of the deprotonated species, B. For
the solution at a pH where both species are present, the chemical shifts of are of intermediate
value. The chemical shifts of are related to the chemical shifts of the protonated and
deprotonated forms by equation 4,
BH xBH B xB
(4)
Where is the observed chemical shift at the specified pH; xBH+ and xB are the mole fraction of
the two species at the same specified pH. To determine the pKa of the pyridinium cation of
interest, xBH+ and xB will be calculated using the chemical shifts using equations 5 and 6.
Equation 5 uses the chemical shift of a specific ring proton to determine the mole fraction of the
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deprotonated species (xB) and equation 6 can be used to determine the mole fraction of the
protonated species (xBH+), since the sum of the two fractions must equal one.
@ lowestpH
xB (5)
@ lowestpH @ highestpH
xBH xB 1 (6)
The pKa can be estimated using equation 7. Equation 7 is equivalent to equation 3 except
that concentrations of the two species have been replaced by mole fractions.
xB
pH pK a log (7)
xBH
Equipment and Reagents:
NMR tubes (~16), pH meter, pH probe and calibration solutions (pH=4 and pH=7),
disposable pipettes, pyridine, 2,6-lutidine, tetramethylammonium iodide, HCl (1M), KOH
(1M), HCl (0.1M) and KOH (0.1M).
Procedure:
1. Preparation of sample solution: In a 250mL beaker dissolve approximately 750
milligrams of sample (assigned) and 100 milligrams of tetramethylammonium iodide in
~50mL of deionized water, mix well. Set up a pH electrode in this solution, set on a stir
plate.
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 4 mL of concentrated HCl (~12 N) to a properly labeled test tube. Then
slowly add about 44 mL of H2O to the test tube and swirl to mix well. To prepare the
~0.1 M solution, dilute 10mL of the ~1.0 M HCl to 100mL.
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 ~ 2.5 g of KOH into a properly labeled test tube. Then, add about 40 mL of H2O
into this test tube, mix well. To prepare the ~0.1 M solution, dilute 10mL of the prepared
~1.0 M KOH solution.
4. Adjusting pH of the sample solution: Take the solution made in step 1 and add HCl
(1M HCl or 0.1M HCl as appropriate) to bring the pH to ~1, mix well, record the pH
value to the nearest 0.01). Fill a labeled NMR tube with the solution of which the pH is
now known.
5. Continue to prepare a series of samples until the pH ~8.5 for pH increments of ~0.5. The
pH of the sample solution can be adjusted by adding the prepared HCl or KOH solutions
drop wise to the reaction 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. The
~1.0 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. ~0.1 M
solutions of HCl and KOH will be used most often when adjusting the pH of the sample
solution at other points. Label each NMR tube before filling it.
6. Acquiring NMR spectra: After the pH of the solutions has been adjusted appropriately,
the samples can be loaded to auto sampler and a spectrum acquired using the NMR
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spectrometer. The routine for acquiring data are such that water (solvent) peaks are
suppressed in all spectra; however a residual water peak will appear.
7. Determination of peak shifts at various pH values: After the spectra have been
collected, the data would be processed automatically (Fourier transform the FID, adjust
the phase, and set the ppm of the reference peak). Determine the peak positions for each
set of equivalent ring protons at the various pH values.
Data analysis:
To process the data collected using the NMR, the J-NMR program will be used. This is set up to
perform all necessary data manipulations; 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 with protons of the
tetramethylammonium ion have a chemical shift assumed at 3.207 ppm. In order to see the
tetramethylammonium proton peak and the sample proton peaks, you may 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 below. After the
x-axis of the NMR spectrum has been correctly calibrateda, the chemical shift for a proton peak
can be determined. 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.
A. Full scale spectrum of water showing the peak at 4.85 ppm (no solvent suppression).
B. Expanded scale of a typical spectrum showing tetramethyl ammonium reference protons at
3.207 ppm and the aromatic ring protons between 7 and 9 ppm.
Record the chemical shift for each set of equivalent ring protons in the sample as a function of
pH. Plot the chemical shift vs. pH for all ring protons (three plots for pyridine and two for 2,6-
lutidine).
For each data point, calculate the mole fraction of base (xB), the mole fraction of acid (xBH+).
Plot pH vs. log[xB/xBH+] for each equivalent set of ring protons, obtain the best fit lines, and
estimate the pKa value and the associated uncertainty. Use the observations from the changing
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chemical shift portion of the curve to determine the pKa of the sample. Compare with the
literature vale.
(in CDCl3) ppm (B) (in CDCl3) ppm
Pyridine H 2,6 Lutidine. (A)
(C) (C)
H H (B)
A 8.593 (B)
A 7.421
B 7.617 B 6.929
(A) H N H (A) C 2.513 H3C N CH3
C 7.231
(C) (C)
Spectral data from
http://sdbs.db.aist.go.jp/sdbs/cgi-bin/direct_frame_disp.cgi?sdbsno=518
http://sdbs.db.aist.go.jp/sdbs/cgi-bin/direct_frame_disp.cgi?sdbsno=688
References
1. National Institute of Advanced Industrial Science and Technology (AIST); Spectral
Database for Organic Compounds (SDBS).
http://sdbs.riodb.aist.go.jp/sdbs/cgi-bin/cre_index.cgi
2. Sawyer, D. T.; Heineman, W. R.; Beebe, J. M. Chemistry Experiments for Instrumental
Methods; John Wiley & Sons: New York, 1984.
