Thin layer chromatography (TLC) is a facile and versatile technique for the separation of compounds in a mixture, including the separation of lipid classes. While it provides excellent resolution, and lipid species can be visualized with staining techniques, the behavior of a separated compound on a TLC plate is usually described in terms its retention factor (Rf), which is consistent only if the chromatography conditions are constant. Further, the Rf values for the compound of interest must be known beforehand for identification. In this work, we couple TLC with continuous Raman detection from the point of sample to the solvent front to investigate phospholipids, which constitute a major component of cell membranes. Various diseases are associated with alterations in composition and levels of phospholipids and coupling TLC for routine separation, along with Raman spectroscopy, which provides high chemical specificity to differentiate lipid species, provides a relatively quick and easy means to probe lipid membranes along with the sterols found within them.<br/>TLC was performed by running either empty or cholesterol-loaded synthetic small, unilamellar vesicles (SUVs) or multilamellar vesicles (MLVs) composed of dioleoylphosphatidylcholine (DOPC) lipids on a solid silica support. Separation was achieved using silica as the stationary phase and a 60:30:4 ratio of chloroform:methanol:water as the mobile phase. Cholesterol and DOPC were used as standards. Plates were visualized with phosphomolybdic acid and heat. Retention factors were calculated for all spots. Next, Raman spectra were recorded from spots on the TLC plates using a custom-built Raman microspectrometer that uses a stabilized 785 nm, 80 mW diode laser. Raman spectroscopy provided detection of vibrational frequencies of specific DOPC and cholesterol functional groups. Next steps include running lipids extracted from the serum of healthy domestic swine using chloroform and methanol to test if coupling these two analytical methods can separate and identify more complex biological samples for applications in medical diagnosis and screening.

Coupling raman spectroscopy with thin layer chromatography to identify lipid membranes

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