Affinity between biomolecules and surface active materials induces the formation of a Langmuir-biofilm (L-biofilm), as the basis for the development of high resolution bioseparation processes. Experiments were performed to characterize the interaction between amphiphilic molecules and proteins, establishing the optimal conditions for bioseparation. In the model L-biofilm system, Bovine Serum Albumin (BSA) was the protein and arachidic acid was the amphiphilic molecule. The L-biofilm formation is promoted by interactions within the subphase (polar groups) and at the interface (non polar groups). The first stage of the process to create the L-biofilm is the migration of BSA from the water subphase towards the air-water interface. This step is followed by a two-step process that includes diffusion and rearrangement. This process has been modeled using a double exponential equation and it is dominated by diffusion, although rearrangement reveals as a faster process. Once the L-biofilm is formed, phase behavior isotherms first show compressible films with areas per molecule larger than the corresponding to pure arachidic acid, due to the penetration of BSA molecules into the acid monolayer. As compression progresses, BSA is squeezed out of the interface although remains attached to the acid in the subphase. The L-biofilm shows hysteresis, in contrast to the behavior of a pure acid L-film. A probable cause for this behavior is the folding of BSA in the L-biofilm upon compression and a slower unfolding during expansion with loss of surface active material in successive cycles of compression-expansion. Regarding the L-biofilm stability, experimental data show a phase of significant film reorganization, due to the presence of BSA, followed by migration of the L-biofilm towards the subphase. A key variable was subphase pH, because it induces changes in the conformational structure of BSA. Changes in structure affect diffusion, rearrangement and solubility and, therefore, L-biofilm formation, structure and composition. Brewster Angle Microscopy studies show the compactness of the L-biofilms, confirming a good level of L-biofilm formation at pH = 3.8 and 5.1 and a rather low formation at pH = 8.2. (C) 2012 Elsevier B.V. All rights reserved.