Phase separated lipid mixtures systems have been of vast interest due to their possible relationship with lipid raft formation which is believed to facilitate certain protein activities and is associated with important biological processes. While a lipid multilayer system serve as an ideal system for high resolution structural studies with X-ray scattering techniques and has mostly been used to obtain an averaged result for pure or mixed systems (e.g., proteins, peptides in membrane), we have found that multilayer systems of phase separated lipid mixtures are actually able to give independent information regarding the coexisting phases due to the demonstrated columnar order of each of the two phases arising from the selective coupling in the third dimension. This finding opens up another dimension of studies for phase separated lipid mixture systems.

In the first part of the dissertation, we describe a quantitative study of cholesterol partition and its condensing effect in phase separated ternary lipid mixtures using X-ray diffraction. The electron density profiles (EDP) of both phases were constructed and a newly invented EDP baseline-scaling model was used to extract the quantitative information of the cholesterol composition and lipid packing.

Next, the effect of close to 100% hydration on the different phases in the phase separated lipid mixture multilayer was studied. Firstly, we developed a high precision method for achieving and controlling hydration close to 100%, and an accurate method for calibrating the relative humidity using a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) multilayer. With this setup, we discovered the novel phenomenon of anomalous swelling type II in the liquid disordered (Ld) phase of the phase separated mixed multilayer. This anomalous swelling is most likely due to the hydrophobic mismatch energy at the phase boundaries.