Both H-1 and P-31 NMR are useful for liposome studies. P-31 is most useful because data interpretation is more straightforward.

If your sample preparation question refers to NMR samples, then for P-31 you need enough D20 to maintain a stable field-frequency lock. The pH should be relevant to the situation you’re interested in. A biological study should use physiological pH. Exclude all other non-lipid molecules with phosphorous. Samples should be fresh. It could be useful to record spectra every hour or so to make sure the lamellarity is stable.

The inside/outside bilayer surface molar ratios can be estimates using simple lanthanide salt shift reagents. P in the outside bilayer

P-31 signal amplitudes are affected by NOE when H-1 decoupling is used. Amplitudes that reflect actual P moles require using gated decoupling. The delay between FIDs should be at least 3 times the longest P-31 T1 estimate which means some simple T1 measurements are needed.

Multilamellar bilayers are complicated to study. Their P-31 line widths and line shapes are sensitive to particle size. Larger multilamellar

Large multilamellar bodies also undergo anisotropic motional averaging. Their line shapes could be asymmetrical due to CSA. A magic-angle spinning , P-31 NMR probe would be a powerful way to estimate lamellarity. The broad, CSA signals from large, asymmetrical multilamellar bodies from would have much narrower, symmetrical line shapes when the sample is spun at the magic angle.

Another difference for multilamellar bodies is the lanthanide shift reagent won’t affect P chemical shifts for lipids that are not directly exposed to the solvent.

A PhD student should know how to do their own literature search. However, if you look at my ReseachGate profile you will find publications from the mid-1970s on phospholipid vesicle NMR studies.

Dear Ayshwarya Ravikumar Like indicated by William C Hutton there are great resources in the (older) literature. See for example the papers by Cullis & de Kruijff like their 'classic'

Cullis, P. T., & De Kruijff, B. (1979). Lipid polymorphism and the functional roles of lipids in biological membranes. Biochimica et Biophysica Acta (BBA)-Reviews on Biomembranes, 559(4), 399-420.

https://www.sciencedirect.com/science/article/abs/pii/0304415779900121

Best regards.

PS. Or papers from the lab of de Kruijff and co-workers

De Kruijff, B., Verkley, A. J., Van Echteld, C. J. A., Gerritsen, W. J., Mombers, C., Noordam, P. C., & De Gier, J. (1979). The occurrence of lipidic particles in lipid bilayers as seen by 31P NMR and freeze-fracture electron-microscopy. Biochimica et Biophysica Acta (BBA)-Biomembranes, 555(2), 200-209.

https://www.sciencedirect.com/science/article/abs/pii/0005273679901603

Nuclear Magnetic Resonance (NMR) spectroscopy offers powerful tools for characterizing liposome lamellarity, with ³¹P NMR being particularly informative due to its sensitivity to phospholipid dynamics and organization. When preparing samples for analysis, liposomes should be suspended in an appropriate buffer such as HEPES or PBS at concentrations typically ranging from 10 to 50 mg/mL lipid content, with careful attention to maintaining temperatures above the phase transition of the lipid components to ensure bilayer fluidity. For unilamellar vesicles, extrusion through polycarbonate membranes of defined pore sizes (commonly 100 nm) helps achieve uniform populations, while multilamellar preparations can be obtained through conventional thin-film hydration methods without extrusion. The ³¹P NMR spectra of unilamellar liposomes characteristically display a narrow, symmetric peak centered around 0 ppm, reflecting the rapid isotropic motion of phospholipids in a single bilayer, whereas multilamellar structures produce broad, asymmetric patterns with chemical shift anisotropy spanning approximately ±15 ppm due to restricted molecular motion in stacked bilayers.

Sample preparation for NMR experiments typically involves the use of Shigemi tubes for small volumes or standard 5 mm NMR tubes with the addition of 10% D₂O for lock signal stabilization, with consideration given to the potential broadening effects of paramagnetic ions that can be mitigated by EDTA addition. Complementary ¹H NMR analysis can provide additional insights through examination of lipid headgroup signals, such as the trimethylamine resonance of phosphatidylcholine around 3.2 ppm, where linewidth differences between uni- and multilamellar preparations reflect variations in molecular mobility. For more complex systems or to resolve overlapping signals, magic-angle spinning (MAS) techniques can be employed to narrow linewidths in multilamellar samples. Key references for methodology include foundational work by Cullis and de Kruijff on chemical shift anisotropy analysis in membrane systems, as well as contemporary protocols described in the Journal of Liposome Research, while data interpretation benefits from established correlations between spectral features and bilayer organization documented in Chemistry and Physics of Lipids. The integration of NMR findings with complementary techniques such as dynamic light scattering for size distribution analysis provides a more comprehensive understanding of liposome structural characteristics. Further refinement of experimental approaches can involve optimization of pulse sequences and relaxation delays to enhance signal resolution, particularly for quantitative comparisons between different lamellar preparations.