MicroSolv Technologies Product Search


	Text Size: A A CombiSep™ pKa Determinations Why use Capillary Electrophoresis (CE) for pKa Determinations? Evaluation of pKa Values by Capillary Electrophoresis The acid dissociation constant (pKa value) of an ionizable compound is defined as the pH value at which the dissociated and undissociated species are of equal equilibrium concentration, and is a fundamental physicochemical property that strongly influences many properties. For example, the neutral form of the compound is less water soluble, more lipophilic, and possesses higher membrane permeability than the ionized form. Experimental knowledge of compound pKa values provides a measure of the extent of compound ionization across the pH range of pharmaceutical relevance, and is highly beneficial for predicting compound absorption, distribution, metabolism, and excretion (ADME) properties. The pKa value also plays an important role in the development of drug delivery formulations. Over the past decade, capillary electrophoresis (CE) has emerged as a valuable tool for the evaluation of compound pKa values, as it possesses many favorable qualities: Potential impurities and degradants can be separated from the target compound Intimate knowledge of sample concentration is not required for analysis Sparingly soluble compounds with a suitable UV chromophore can be analyzed No changes in spectral properties are required for detection of a pKa value Minimal sample amounts are required for analysis (µg amounts) Numerous publications have appeared describing the use of CE for pKa analysis including a recent review. Several of these references are listed on this website. Potential users are encouraged to study these articles to improve their knowledge of CE-based pKa analysis, and best understand the strengths and limitations of the technique. To determine the pKa value(s) of a compound by CE, it is necessary to measure the migration times of the compound in relation to a neutral marker (usually DMSO) over a range of pH values. From the migration times of the compound (t_a) and neutral marker (t_m) in seconds, the effective mobility (µ_eff) can be calculated via Equation 1: Eqn 1 µ_eff = L_dL_c/V (1/t₀-1/t_m) Where L_d is the length of the capillary to the detector, L_c is the capillary total length (in cm), and V is the applied voltage (V). A plot of µ_eff vs pH yields a sigmoidal-shaped titration curve, from which the inflection point(s) define the pKa value(s). Using nonlinear regression analysis a best-fit line can be applied to the data using a set of standard fitting equations depending on the number of ionizable groups, and the pKa value determined. Equations used for the evaluation of up to three ionizable groups have been described in the literature and can be viewed on this site.

[TOP]