Higher order equilibria and their effect on analyte migration behavior in capillary electrophoresis

Area of Science:

• Analytical Chemistry
• Separation Science
• Physical Chemistry

Background:

• Capillary electrophoresis (CE) is a powerful separation technique.
• Analyte-additive interactions, particularly complexation, significantly influence migration behavior.
• Understanding higher-order binding stoichiometries (1:1 and 1:2) is crucial for accurate CE analysis.

Purpose of the Study:

• To quantitatively investigate the impact of 1:1 and 1:2 analyte-additive interactions on analyte migration in CE.
• To derive and validate equations accounting for first- and second-order equilibria in CE.
• To compare binding isotherms and assess the utility of capacity factors for analyzing complex equilibria.

Main Methods:

• Derivation of equations based on individual capacity factors for 1:1 and 1:2 binding.
• Quantitative analysis of analyte migration behavior using microscopic equilibrium constants and mobilities.
• Experimental validation using 4,4'-biphenol, 4-phenylphenol, and phenol with hydroxypropyl-β-cyclodextrin (HP-β-CD).

Main Results:

• Developed equations accurately describe analyte migration influenced by mixed 1:1 and 1:2 binding stoichiometries.
• Demonstrated the calculation of higher-order equilibrium constants and complex mobilities from binding isotherms.
• Showcased the advantage of using capacity factors in CE for resolving complex interactions and optimizing experimental conditions.

Conclusions:

• The derived equations provide a robust framework for understanding and predicting analyte migration in CE with complex binding.
• Accurate determination of equilibrium constants and mobilities is achievable even with higher-order interactions.
• This work enhances the quantitative application of CE for analyzing complex molecular interactions.