The membranes in this condition would split with their hydrophobic side in the air-water interface and their hydrophilic side in the water. By compressing the surface film and measuring the energy it took to compress, they found that the energy increased greatly when the film was about 2X the area calculated for the cell surfaces. They deduced that the cell membrane must be a bilayer. Freeze etching has also shown that when the membrane is split in the middle that proteins are not equally distributed on the two sides. Some proteins seem to float on one surface or the other and other proteins seem to penetrate the membrane giving rise to the concept of a fluid mosaic model .
Some proteins are fixed at certain points in the membrane, others are movable.
Electron micrographs of mitochondrial inner membranes indicate a regular, organized protein distribution across the inside surface. Protein digestion will remove these projections. The regularity of the protein structures hints at the requirement for physical position to insure function. The diffusion rate varies inversely with distance , and directly with temperature , concentration gradient , area through which diffusion takes place, and a fudge factor called the diffusion coefficient . The diffusion coefficient is empirically determined and varies with molecular size along with other attributes and solvent characteristics.