Protein separation during ion-exchange chromatography implies complex physicochemical events. This work has evaluated the chromatographic behaviour of a complex cell proteome on commercial agarose-based adsorbents. Various ligand types in the cation- and anion-exchange mode were studied. ANX-Sepharose, a weak anion exchanger, performed similarly to the strong anion exchanger-type materials. Proteomic tools were applied in order to understand protein separation. Experimental evidence showed a correlation between apparent isoelectric point distributions and the mobile phase conductivity. Molecular weight distributions were unaffected by the elution position. On the basis of two-dimensional electrophoresis, operational windows were described having typical minor contaminants. These could be annotated for future implementation of in silico downstream processing.