Methods for Measuring pKa
UV-metric methods provide pKa results for samples with chromophores whose UV absorbance changes as a function of pH. A chromophore is a region of a molecule such as a series of conjugated double bonds that absorbs UV light. About 70% of samples have UV-active pKas.
On SiriusT3, the Fast UV method typically requires 5μL of a 10mM solution of sample, and measures absorbance at 250 wavelengths and 54 pH values in a buffered solution in about 5 minutes. A slower method in unbuffered solution extends the pH range below 1 or above 13. The 3-D graph shows data for the measurement of Labetalol pKas. The other graphs are 2-D projections showing change in absorbance vs. pH and vs. wavelength, with percent species and molar absorbance coefficients overlain.
In this example, the acidic phenol (highlighted in red) is part of a conjugated region of the molecule with strong UV absorbance, and this allows the pKa of 7.35 to be determined. The basic amine (highlighted in blue) is not part of a chromophore. Even so, small changes in absorbance at low wavelength allow the amine pKa of 9.08 to be determined. These changes may have arisen because the molecule is flexible, and the amine influences the absorbance of the ring on the right. With its multiwavelength capability, SiriusT3 can measure pKas of many molecules for which pH change induces only weak spectral changes.
3-D spectra for Labetalol deconvoluted inot Absorbance vs. pH and Absorbance vs. Wavelength
pH-metric methods are based on potentiometric acid-base titration. On SiriusT3, they typically require about 0.5 mg of sample. Results are obtained by a complex computational process. The pH of each point in the titration curve is calculated using equations that contain pKa, and the calculated points are fitted to the measured curve by manipulating the pKa value. The pKa that provides the best fit is taken to be the measured pKa.
pH-metric methods will measure all pKas between 2 and 12, provided the sample is in solution throughout he experiment.
In this example the pKa of propranolol has been measured, with a result of 9.53.
Measured data for Propranolol, with poorly-fitted and well-fitted calculated curves
pKa of poorly soluble samples is determined by measuring psKa values in water-solvent mixtures by UV-metric or pH-metric methods, and extrapolating to aqueous conditions.
Three titrations are done consecutively in the same vial. Yasuda-Shedlovsky is the standard extrapolation method, in which the X-axis plots the inverse of the dielectric constant of the water-solvent mixture at the experimental percentage of solvent. This example shows pKa of Fluoxetine determined from three UV-metric titrations in methanol-water.
The pKa is equivalent to the intercept at 0% solvent minus log[H2O] (1.75 for pure water), providing a result of 9.88 for this sample.
When Sirius was founded in 1990, no instrument was available for determining pKa values from potentiometric titration curves or pH-UV data. Although the classical techniques were understood, the measurement of pKa values of drugs was a highly specialised task. We introduced software and dedicated instrumentation that could handle small quantities of sample, and cosolvent methods for poorly soluble samples. A large number of pharmaceutical companies, universities and agrochemical companies now use our instruments, and we believe we provide the de facto worldwide standard for pKa measurement.
Sirius instruments measure pKa values by potentiometric titration and spectrophotometric titration. These classical techniques have been used for many years for pKa measurement. They were outlined in OECD Guideline 112 for the measurement of Dissociation Constants (pKa values) in Water which was adopted in 1981, and described in a widely-accepted monograph (Albert, A.; Serjeant, E. P., The Determination of Ionisation constants. Chapman and Hall, London, 2nd Edition 1984) that we used during development of our technology.
Sirius-measured pKa values feature in many publications, some of which are listed in our bibliography. Of these, the following were validation studies that compared our pKa results with independently-measured pKa values:
20 compounds compared: J. Comer, K. Chamberlain and A. Evans. Validation of pH-metric technique for measurement of pKa and logPow of ionizable herbicides. SAR QSAR Environ. Res., 1995, 3, 307-313
25 compounds compared: K. Takács-Novák, K. J. Box and A. Avdeef. Potentiometric pK(a) determination of water-insoluble compounds: Validation study in methanol/water mixtures. Int. J. Pharm., 1997, 151, 2, 235-248 25 compounds compared: K. Y. Tam and K. Takács-Novák. Multiwavelength Spectrophotometric Determination of Acid Dissociation Constants: A Validation Study. Anal. Chim. Acta, 2001, 434, 157-167
71 compounds compared: K. Box, C. Bevan, J. Comer, A. Hill, R. Allen and D. Reynolds. High-throughput measurement of pKa values in a mixed-buffer linear pH gradient system. Anal Chem, 2003, 75, 4, 883-92
The graph belowcompares 32 pKa values measured on SiriusT3 with published measured pKa values.