Determination of Thermodynamic Binding Constants by Affinity Capillary Electrophoresis

CARLINA LANCIONI; SONIA KEUNCHKARIAN; LEONARDO G. GAGLIARDI

Mendoza

Simposio; 24th Latin-American Symposium on Biotechnology, Biomedical, Biopharmaceutical, and Industrial Applications of Capillary Electrophoresis and Microchip Technology; 2018

Determinationof equilibrium constants (association/dissociation) is a relevantsubject in different disciplines since its knowledge is useful formany application fields. A wide variety of instrumental techniqueshave been employed for determining equilibrium constants. However,affinity capillary electrophoresis (ACE) has undoubtedly become oneof the most popular and has been positioned as a useful tool to studychemical equilibria. Different ACE approaches have been reviewed inseveral publications [1-5]. Nonetheless, although additionalcorrections are usually taken into account, it should be noted thatmeasured constants are stoichiometric or apparent, not truethermodynamic constants, i.e.they are only valid for the particular experimental conditions inwhich they were determined. Rigorous works must be based onthermodynamic equilibrium constants, and their determinations requireconsidering the activities of the individual forms involved.Inthis study, a strategy to determine thermodynamic binding constantsby ACE is presented. In order to simplify mathematical treatment,analogy with the well known acid-base dissociation equilibrium isused: instead of ligand concentration [X],negative logarithm of ligand concentration (or activity), pX= -log[X],is used. This approach allows to establish criteria to perform anexperimental design, whether the goal is to determine bindingconstants, or to optimize analytical separations. In addition, themethod provides free analyte and analyte-ligand complexelectrophoretic mobilities, even when binding constants are low andthus, the complexed analyte fraction is also low. This is useful as abase to rationally analyze a diversity of situations, i.e., for allpossible analyte-ligand charge combinations.Experimentally,enantiomeric ACE separation based on the use of chiral selectors wasaddressed. 2-hydroxypropyl-β-cyclodextrin was chosen as a modelligand, and both enantiomeric forms of four pharmaceutical drugs(propranolol, pindolol, oxprenolol and homatropine methylbromide)were considered as model analytes. Thermodynamic constants for allcomplexes, as well as the actual mobilities of both free andcomplexed analytes were obtained.References1.K.L. Rundlett, D.W. Armstrong, J. Chromatogr. A.721 (1996) 173?186.2.Y. Tanaka, S. Terabe, J. Chromatogr. BAnal. Technol. Biomed. Life Sci. 768 (2002) 81?92.3.K. U?elová-Včeláková, I. Zusková, B. Ga?, Electrophoresis. 28(2007) 2145?2152.4.Z. Chen, S.G. Weber, Trends Anal. Chem. 27 (2008) 738?748. 5.C. Jiang, D.W. Armstrong, Electrophoresis. 31(2010) 17?27.p { margin-bottom: 0.25cm; direction: ltr; line-height: 120%; text-align: left; }a:link { color: rgb(0, 0, 255); }