Preparation Method Determines Valinomycin Organization and Phase Behavior in Charged Langmuir Monolayers

Amphiphilic molecules are typically incorporated into lipid Langmuir monolayers by two distinct methods: co-spreading, where lipid and amphiphile are deposited simultaneously, and injection, where the amphiphile is introduced into the aqueous subphase beneath a preformed monolayer. Whether these methods produce equivalent molecular arrangements remains an open question. Using valinomycin as a model ionophore, we investigate this question across lipid monolayers bearing anionic, zwitterionic, and cationic headgroups. Surface pressure-area isotherms, fluorescence imaging, and vibrational sum-frequency generation spectroscopy together reveal that the two preparation methods produce fundamentally different molecular organizations. Cospreading yields a uniform, well-incorporated monolayer, while injection produces a kinetically trapped, spatially heterogeneous state in which valinomycin resides predominantly at the headgroup-water interface. The magnitude of these differences scaled with lipid headgroup charge, implicating specific interactions between lipid headgroups and the carbonyl-lined cavity of valinomycin as key determinants of the incorporation pathway. These findings establish that preparation history is a critical parameter governing molecular organization in mixed amphiphile-lipid monolayers.