Investigation of plateau methods for adsorption isotherm determination in supercritical fluid chromatography

•Isotherm determination methods based of overloaded profiles works well for SFC.•However, the Perturbation Peak isotherm determination method fails for SFC.•Adsorption Energy Distribution calculations was shown to work also for SFC.•The reason for Perturbation Peak method's failure was investigated and explained.•The plateau method, frontal analysis, seems to fail for the same reason.

The Perturbation Peak (PP) method and Frontal analysis (FA) are considered as the most accurate methods for adsorption isotherms determination in liquid chromatography. In this study we investigate and explain why this is not the case in Supercritical Fluid Chromatography (SFC), where the PP method does not work at all, using a modern analytical system. The main reason was found to be that the solute to be studied must be dissolved in the MeOH reservoir before it is mixed with CO2. Since the solute occupies a certain partial volume in the reservoir, the larger the solute content the larger this fractional volume will be, and the final MeOH fraction in the mobile phase will then be smaller compared to the bulk mobile phase without solute in the modifier. If the retention of small injections on the concentration plateaus, i.e., “analytical-size” perturbation peaks, is sensitive to small variations of MeOH in the eluent, this will seriously decrease the accuracy of the PP method. This effect was verified and compensated for and we also demonstrated that the same problem will occur in frontal analysis, another concentration plateau method.