Gaining insight into the relative positions of each component within lipid nanoparticles (LNPs) is essential. Such knowledge can help clarify the intricate behaviour of LNPs and contribute to the design of more efficient and safer formulations. Furthermore, it can play a vital role in quality control during the scale-up of manufacturing processes, thereby enhancing the translation of LNPs from the laboratory to clinical applications. Examining the Dlin-MC3-DMA profile suggests that it is located at the surface, with molecular and fragment ions exhibiting a drop in signal intensity from the surface (Figure ). This is mirrored by fragments from all parts of the molecule, headgroup and fatty acid tails. Towards the core, these same ion intensities begin to increase, suggesting that it is also at an elevated amount in the core of the particle. Notably, the depth in the profile where the intensity decreases is at the same position that the fatty acid intensities increase in the DMG PEG molecule, suggesting the presence of a potential hydrophobic barrier layer that could be envisaged as separating molecules like the ionisable lipid from the particle core. Molecules will partition based on their molecular properties, such as charge state and hydrophilicities, as exhibited at the pH of particle formulation in the formation of this hydrophobic barrier layer. As the molecular assembly of LNPs contributes to the determination of LNP structure and thereby function, further experimentation systematically varying the pH of formulation could enable the assessment of this hydrophobic barrier and its impact on molecular distribution.