Estimation of Nonlinear Adsorption Isotherms in Gradient Elution RP-LC of Peptides in the Presence of an Adsorbing Additive.

ABSTRACT: In electrostatic repulsive interaction chromatography, using a charged surface hybrid sorbent carrying positive charges can improve the peak shape of peptides in reversed-phase liquid chromatography (RP-LC), especially in overloaded conditions, compared with standard C18 sorbents. However, the positive surface charges can interact with anionic additives commonly used in peptide separations, e.g., trifluoroacetic acid (TFA), complicating adsorption isotherm estimation. We investigated how the competition for available adsorption sites between TFA and two peptides influenced the adsorption isotherm in gradient elution. A model accounting for the competition with TFA was compared with a model neglecting TFA adsorption. We found that the two models predicted elution profiles with the same accuracy. We also found that the adsorption isotherms were extremely similar in shape, leading to the conclusion that neglecting the competition with TFA is a valid approximation enabling faster and more robust adsorption isotherm estimation for the studied type of sorbent.