pKa

pH-metric pKa assays

An advantage of Sirius pH-metric methods is that they can be used for pKa values between 2 and 12 for any ionizable compound, even those that have no pH-active UV chromophore such as aliphatic amines and carboxylic acids. A weakness is the relatively large weights of sample required to effect a measurable pH change (e.g. 2-5mg), which can be a problem if not enough sample is available, or if the weight required does not dissolve in water-solvent mixture. (These problems are overcome using D-PAS for pH-UV methods). Because of the need to achieve pH equilibration after each addition of titrant in unbuffered solution, pH-metric experiments typically take 20 to 60 minutes per titration. Three titrations in different water-solvent mixtures followed by Yasuda-Shedlovsky extrapolation will be required to measure water-insoluble samples.

pH-UV assays

Sirius standard D-PAS methods for pH-UV measure pKa in unbuffered solutions (aqueous or water-solvent). Typical experiments take 20-30 minutes per titration. They can be used to measure pKa values between 1 and 13 of any ionizable group associated with a UV-active chromophore. Concentrations required for Standard D-PAS methods are very low, requiring sample weights of no more than 0.2mg. Three titrations in different water-solvent mixtures followed by Yasuda-Shedlovsky extrapolation will be required to measure water-insoluble samples. Standard D-PAS methods can be used to measure pk and kz microconstants of zwitterions and ampholytes. A weakness of Standard D-PAS methods is that each sample requires a reference spectrum measured in a separate vial, so no more than 25 samples can be measured in a run.

Optimal pKa measurement

Because of the great diversity of structures of drug molecules, it is difficult to find a single method that can be relied on to measure any and all pKas in just one attempt. An efficient approach is first to use the Fast D-PAS technique in aqueous solution. This 4-minute experiment will often provide a valid result, but if it does not, the reason for failure will allow the analyst to choose an appropriate alternative method which will work. Fast D-PAS is a hybrid pH-metric/UV technique in which a small weight of sample (typically 0.2mg in DMSO stock solution) is dissolved in a multi-component buffer solution and titrated between pH 2 and 12. The presence of the buffer allows for a stable pH to be reached very quickly after each addition of titrant. The result is obtained from changes in multiwavelength UV spectra with respect to pH. It is obvious from the data quality if the Fast D-PAS method works, and this data quality can be automatically monitored by software without need for manual inspection. If the method works, then the result will be the aqueous pKa, which is the result required. Moreover, the result will have been obtained in about 4 minutes.

There are two principal reasons why the Aqueous Fast D-PAS technique could fail: the sample could precipitate during the assay, or it could have no pH-active chromophore. If the sample has UV response but it precipitated, it can be re-run by Fast D-PAS (or standard D-PAS) in the presence of cosolvent. If it had no UV response but it did not precipitate, it can be re-run by a pH-metric method under aqueous conditions. If it had no UV response and it precipitated, it can be re-run by a pH-metric method in the presence of cosolvent.

Both precipitation and lack of UV response can be can be automatically deduced from Fast D-PAS data. In principle, there is no need to know the molecular structure for Fast D-PAS or pH-metric methods, although data interpretation and understanding are greatly enhanced if the structure is available. For example, data interpretation might be difficult for a molecule with two pKas, only one of which is UV-active, or for a molecule with an extreme (i.e. very low or high) pKa (e.g. below 1, above 13). Extreme pKas cannot be measured by Fast D-PAS or pH-metric methods. However, they are accessible by standard D-PAS methods done in unbuffered solutions.

The Fast D-PAS technique was used to measure pKa values of two very insoluble compounds (glipizide and levothyroxine) in Sköld et al's recent study of 24 compounds chosen for assay validation. These compounds were measured in aqueous solution because they formed supersaturated solutions when converting from their ionised to unionised forms, and did not have time to precipitate in a short experiment. Because the Fast D-PAS experiment takes only a few minutes, pKas of labile samples can be measured with less risk they will decompose under acid or base conditions.

High throughput pKa measurement.

For unattended measurement of pKa of up to 384 samples in one run, it's hard to beat the throughput of Sirius ProfilerSGA. All samples are measured under identical conditions e.g. all aqueous, or all at a fixed water-solvent ratio. Results are calculated by ProfilerRefine3 software, which all-at-once processes data for all samples in a 96-well plate, analysing the data quality using a "traffic lights" system to alert users to questionable results. ProfilerSGA is recommended if high throughput with minimum user intervention is required. However, it is less versatile than the alternative Fast D-PAS methods on GLpKa.

Fast D-PAS uses built-in reference spectra so that all 50 vials can be used for samples. Each of the 50 samples can be run under appropriate conditions, e.g. all aqueous (though some may precipitate), or all at one fixed water-solvent ratio. Alternatively each sample can be run at three different ratios of water-solvent, with result obtained by Yasuda-Shedlovsky extrapolation. Alternative methods are always available to study interesting or difficult samples.