Effects of heterogeneity on the characterization of chromatographic stationary phases

An important aspect of high-performance liquid chromatography (HPLC) is the characterization of the stationary phase in the column. Frontal analysis and zonal elution are two experimental methods used for characterizing the binding properties and the binding capacities of a variety of HPLC columns in both industrial and academic laboratories. Most data obtained is interpreted as if the stationary phase in the column was homogeneous. However, there are numerous studies that demonstrate that many different types of stationary phases are actually heterogeneous.; This study examined how the binding capacities and equilibrium constants measured by frontal analysis and zonal elution are affected by ligand heterogeneity in affinity columns. Equations derived for n- and two-site systems gave good agreement with results obtained for the binding of L-thyroxine and the binding of (R)-warfarin to coupled columns containing human serum albumin or pigeon serum albumin. The same equations were used to examine how different degrees of ligand heterogeneity affected the apparent binding capacities or equilibrium constants measured using the linear range of double reciprocal frontal analysis plots or single reciprocal zonal elution plots. A large proportion of two-site systems gave good estimates (i.e., less than 10-20% error) for the true column capacity and the association constant of the highest affinity ligand in the column. A smaller, but still appreciable, fraction of all three and four-site cases also produced good estimates of these values when using frontal analysis. The results of this work are not limited to protein-based affinity columns but should be applicable to any type of stationary phase that has well-defined binding regions and relatively fast, reversible interactions with solutes.