Low-frequency Raman spectroscopy as a new tool for understanding the behaviour of ionisable compounds in dispersed mesophases

Hypothesis: Low-frequency Raman (LFR) spectroscopy is proposed as a novel non-destructive methodology to probe pH-related phase transitions in self-assembled lipid particles. In this case, dispersed lipid mesophases were composed of ionisable oleic acid (OA) or nicergoline (NG) in monoolein (MO). The sensitivity of LFR spectroscopy to low-energy intermolecular vibrations was hypothesised to be due to structural transformation in ionisable dispersed mesophases upon changes in pH. Method/experiment: Phase transitions of dispersed mesophases of MO mixed with OA or NG were induced by varying the pH of the aqueous buffer. The structural transformations were studied using LFR spectroscopy, recording the corresponding changes in the vibrational density of states (VDOS) upon changes in pH and analysed using principal component analysis (PCA). The results were correlated with structural transitions observed in simultaneous small-angle X-ray scattering (SAXS) measurements. Findings: The intensity of the VDOS signal of MO + OA mesophases scaled with phase-specific transformations, such as from the bi-continuous cubic Im3¯m phase (V₂) or lamellar-based vesicles to the reversed hexagonal p6m phase (H₂). For NG subtle changes in the lattice parameter of the V₂ phase of NG + MO mesophases coincided with the apparent dissociation constant (pK_a^app) of NG, however, slight variations between the pK_a^app of NG determined by equilibrated samples analysed using SAXS and non-equilibrated samples analysed using LFR suggest structural hysteresis upon changes in the protonation state of NG. This approach offers an efficient method for studying the phase behaviour of lipid systems under varying pH and potentially other conditions such as temperature.