Application of the split-peak effect to study the adsorption kinetics of human serum albumin on a reversed-phase support.

The adsorption step of human serum albumin on a reversed-phase support was analyzed by studying the "split-peak" effect in mass-overload conditions. This behavior is characterized by the occurrence of a first non-retained fraction and is described by an analytical expression in the case of a Langmuirian adsorption isotherm. The method was applied to determine the column loading capacity, the number of mass-transfer units and the apparent adsorption rate constant measured at a given flow-rate. The nature of the organic modifier influences the split-peak effect: it increases with the eluotropic strength of the organic solvent added to the buffer. Compared to the results with pure buffer, it is the association of two effects, the decrease of the column loading capacity and that of the apparent adsorption rate constant, which increases the split-peak effects observed when methanol and 2-propanol are added to the eluent. These results allow us to gain a better understanding of the role of the organic solvent in the elution behavior of proteins in reversed-phase high-performance liquid chromatography.