This work concerns the successful enantiomeric separation of pyroglutamic acid derivatives, known to be P2X7 receptor antagonists, achieved by electrokinetic chromatography. After a broad screening, two negatively charged cyclodextrins, sulfobutylether--cyclodextrin (SBE--CD), and highly sulfated--cyclodextrin (HS--CD) were chosen as stereoselective agents to cooperate with the BGE for complexation. A fused silica capillary coated with polyethylene oxide, filled with a phosphate buffer (25 mM, pH 2.5) containing various concentrations of CD, was used. Assuming a 1:1 stoichiometry, calculations of the binding constants, employing the three different linearization plots, were performed from the corrected electrophoretic mobilities values of the enantiomers, at different concentrations of SBE--CD and HS--CD in the BGE. The highest complexation was found with the SBE--CD. Among the three equations, results showed better linearity (R-2 > 0.99) using the y-reciprocal fit. This plotting method was then performed to determine the binding constants of each enantiomer at different temperature for compounds 1 and 2 with SBE--CD and HS--CD in order to access to the thermodynamic parameters of the eight complexes. The linearity of the Van't Hoff plot, in the range of 288-303 K leading to negative enthalpy values, showed that the complexation phenomenon is enthalpically controlled and thermodynamically favored.