Surface area exclusion chromatography was used to investigate the adsorption characteristics of the highly branched polyethyleneimine (PEI) molecule and of a related molecule resulting from the grafting of PEI with C12 to C22 alkyl chains. The interfacial relaxation and surface affinity of the two polymers was determined in homogeneous and heterogeneous layers. The presence of hydrophobic moieties within the branched morphology of the grafted PEI molecule was found to modify the adsorption histogram as compared to bare PEI and to lead to greater interfacial stability. The relaxation of the bare and grafted macromolecules proved to be of equal extent but to develop at different rates within homogeneous layers. In heterogeneous layers composed of the two polymers, the slower relaxation of the grafted macromolecules decreased the rate of relaxation of the bare molecules, while the faster relaxation of the bare molecules strongly increased the rate and extent of the relaxation of the grafted macromolecules. The same technique was then used to determine the mode of establishment of the layers. The polymer coating profiles on successive glass fiber filters of the chromatography column were found to depend on the sequence of injection of the two polymers. Simultaneous and sequential adsorption processes were analyzed on the basis of the random sequential adsorption of macromolecules.