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pKa and log P

Understand the physicochemical properties of your compound by using our pKa and log P service.

The determination of pKa and log P is offered within Cyprotex's in vitro experimental services. Cyprotex deliver consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.

The measurement of pKa and log P

  • The pKa is the pH at which the molecule is 50% protonated.
  • Log P (or partition co-efficient) is a measure of the lipophilicity of a compound.
  • Cyprotex's pKa and log P determination uses UV-metric and pH-metric technology developed by Sirius. This is considered to be a ‘gold standard’ method for determining these properties.
  • In UV-metric methods, pKa is measured by analyzing changes in multi-wavelength UV spectra during acid-base titration of the sample. UV-metric pKa methods work for compounds with pH-sensitive chromophores.
  • In pH-metric methods, pKa is measured by titrating a solution of the sample in water or solvent with acid and base, and calculating the pKa from the shape of the titration. pH-metric methods work for any ionizable compound, but require more sample than UV-metric methods.
  • The pH-metric method is also used to measure log P in a two-phase acid-base titration in the presence of octanol.
pKa affects solubility, permeability, log D and oral absorption by modulating the distribution of neutral and charged species.

1Di L and Kerns EH. (2003) Current Opinion in Chemical Biology 7; 402-408


pKa and log P protocol

Method Fast UV titration for pKa
UV-metric titration for pKa
Potentiometric (pH-metric) titration for pKa and log P
Instrument SiriusT3
Test Article Requirements per Titration 3-5 µL of 10mM stock solution (UV-metric)
1 mg solid compound (pH-metric)
Partition Solvent used for log P n-Octanol (others available on request)
Data Delivery pKa
log P (optional)
Standard error
Calculated log D at pH7.4 (based on pKa and log P)


Data from Cyprotex's pKa and log P assay

pKa measurements are determined using the SiriusT3 instrument from Sirius-Analytical using either a UV-metric or pH-metric approach.


Figure 1
UV-metric method for measuring pKa values.

UV-metric methods provide pKa results for samples with chromophores whose UV absorbance changes as a function of pH.

On SiriusT3, the Fast UV method measures absorbance at 250 wavelengths and 54 pH values in a buffered solution in about 5 minutes. The slower UV-metric method in unbuffered solution extends the pH range below 1 or above 13. The 3D graph shows data from the measurement of labetalol pKas. The other graphs are 2D projections showing change in absorbance vs. pH and vs. wavelength, with percent species and molar absorbance coefficients overlain.
Figure 2
pH-metric method for measuring pKa values.

pH-metric methods are based on potentiometric acid-base titration. 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 the experiment.
Figure 3
pH-metric method for measuring log P values.

In the pH-metric method for log P, a weighed sample is dissolved in a two-phase water-octanol system, and titrated over a pH range (typically 2 to 12 for bases and ampholytes, 12 to 2 for acids). Although the solution becomes opaque during stirring, the pH electrode continues to measure pH of the aqueous component of the solution. 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 P, and the calculated points are fitted to the measured curve by manipulating the P value. The P that provides the best fit is taken to be the measured P value, which is reported as the logarithm, i.e. log P. As well as log P, the log D value as a function of pH is determined from the data.


Questions and answers on pKa and log P determination

Why is the pKa important?

The pKa of a molecule predicts the degree of ionization the molecule will have at a particular pH.

Most drugs are weak acids or weak bases and exist in solution as an equilibrium between unionized and ionized forms. The ionization potential of a compound affects the distribution of the chemical in solution and affects the availability of the chemical to enter into physical, chemical and biological reactions. According to the pH partition hypothesis, only unionized nonpolar drugs penetrate the cell membrane, and at equilibrium, the concentrations of the unionized species are equal on both sides. The pKa of a compound influences properties such as logD and solubility as well as the absorption, distribution, metabolism, elimination and potency of a compound. pKa can be used in conjunction with other in vitro parameters to predict the pharmacokinetics of a compound using the simulation software, Cloe® PK.

How does pKa affect other pharmacokinetic parameters?

Solubility – Acidic compounds tend to be more soluble at high pH values, and basic compounds tend to be more soluble at low pH values.

Permeability – Acidic compounds tend to be less permeable at high pH and basic compounds tend to be less permeable at low pH.

Metabolism – Electrostatic interactions are determined by the pKa of a compound. These interactions can affect binding of the compound to the active sites of enzymes. For example, nitrogen containing bases where the basic nitrogen is 5-7Å from the site of metabolism has been shown to be important in the metabolism of compounds by CYP2D62.

Protein binding – Binding of drugs to plasma proteins tends to be by hydrophobic and electrostatic interactions. Typically, acidic compounds with moderate lipophilicity are more likely to bind to serum albumin whereas basic compounds with moderate lipophilicity are more likely to bind to α1-acid glycoprotein3.

Excretion – Urinary pH is an important factor in the excretion of a drug. For example, acidic drugs are ionized at alkaline urinary pH and basic drugs are ionized at acidic urinary pH. Only unionized compounds in the tubular fluid will be reabsorbed by passive diffusion4.

How does lipophilicity influence pharmacokinetic properties of a drug?

LogP (partition coefficient) is a measure of the lipophilicity of a compound.

Lipophilicity is a key determinant of the pharmacokinetic behavior of drugs. It can influence distribution into tissues, absorption and the binding characteristics of a drug, as well as being an important factor in determining the solubility of a compound.

How much compound will I need to measure pKa and log P?

Although smaller weights are generally required for pKa and log P assays on the SiriusT3, we normally ask customers to send at least 5 mg of solid compound per parameter measured. If only pKa is required, and if it can be measured by a UV-metric technique, then aliquots of sample in stock solution (e.g., 10 mM in DMSO) can be used.

What are the limits to the pKa and log P you can measure?

pH-metric pKa: The standard range is 2.5 to 11.5. Lower or higher pKas can be measured if the sample is sufficiently soluble.

Fast UV pKa: 2.0 to 12.0

UV-metric pKa: 1.0 to 13.0

pH-metric log P: -0.5 to +5.5. The higher limit may be reduced for bases with pKas below 4 or acids with pKa above 10.

Samples must be in solution during pKa measurement. Turbidity is monitored throughout pH-metric pKa titrations to give warning of precipitation. If the compound is insoluble then it may be necessary to perform the measurements either at a lower concentration or in the presence of a co-solvent. Knowledge of the aqueous solubility of the sample helps in designing the experiment and avoids unnecessary use of the compound in repeat experiments. Information regarding compatible organic solvents is also useful.

How do you measure the pKa values of poorly soluble samples?

pKa of poorly soluble samples is determined by measuring psKa values in water-solvent mixtures, 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.

Do I need to provide the structure of the compound?

Providing the structure is preferable as it helps design the experiment (i.e., pH range and direction of the titrations and weight of compound). It also helps in the data refinement process and interpretation of the data. If the structure is not provided then information on functional groups is required.

Why do you need to measure the pKa of my compound before you can determine the log P?

When samples are titrated in the two-phase water-octanol system on the SiriusT3, the data generated comprises a set of pH readings vs. volumes of titrant. The log P is determined after calculating each pH value to simulate those measured. One of the parameters required in these calculations is the pKa value.


1 Di L and Kerns EH. (2003) Current Opinion in Chemical Biology 7; 402-408
2 Lewis DF. (2000) Biochem Pharmacol 60; 293-306
3 Urien S et al. (2001) In Pharmacokinetic optimisation in drug research Ed. Testa et al 189-197
4 Timbrell JA. (1991) In Principles of Biochemical Toxicology 65-67 

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