SPLIT-PEAK PHENOMENON IN NONLINEAR CHROMATOGRAPHY .2. CHARACTERIZATION OF THE ADSORPTION-KINETICS OF PROTEINS ON REVERSED-PHASE SUPPORTS

The kinetics of adsorption of human serum albumin (HSA) on a reversed-phase support was studied by analyzing the "split-peak" effect in mass-overload conditions. This effect occurs when a fraction of solute is eluted as a nonretained peak. It can be conveniently characterized by calculating the fraction of unretained solute obtained from cumulated injections. The experimental data were fitted with a model that assumes irreversible adsorption and Langmuir second-order kinetics. Its analytical expression, relating the nonretained fraction to the sample size and the flow rate, is a function of both the number of transfer units (related to the apparent adsorption rate constant k(a)) and the maximum loading capacity of the column Q(x). From zonal elution experiments the method permits the determination of the parameters k(a) and Q(x), characterizing the adsorption of HSA on the reversed-phase support. The influences of acetonitrile concentration and temperature on the split-peak effect were examined. In pure buffer, the number of transfer units is equal to 16 at 25-degrees-C. It is about 5 times as large as when acetonitrile is added to the eluent. An important decrease of the maximum loading capacity (Q(x)) is observed in the presence of acetonitrile in the mobile phase. This study gives information about the interaction of free protein with both the support and the solvent.