Together with proteins, carbohydrates and nucleic acids, lipids are designated as macromolecular biomolecules — substances essential to one or more fundamental biological processes. In biological systems, lipids may store energy, act as structural components (as in the phospholipid bilayers of cell membranes), or be involved in intra- and intercellular signaling.

Like other biomolecules, lipids encompass a variety of molecules of diverse structure, chemical behavior and function:

  • Monoglycerides
  • Diglycerides
  • Triglycerides (fats and oils)
  • Fatty acids
  • Waxes
  • Sterols
  • Fat-soluble vitamins (e.g., vitamins A, D, E, K)
  • Phospholipids

Note that “fat” and “oil” are non-technical terms for the same type of triglyceride lipid, differing only in their physical states at room temperature: fats are solids, oils are liquids.

Purification and Analysis of Lipids

In highly heterogenous sample material, lipids are often found forming complexes with other biomolecules such as polysaccharides (glycolipids) and proteins (lipoproteins). Releasing these bound lipids from non-lipid components can be done through acid hydrolysis, e.g., by extended heating in the presence of a relatively high concentration of hydrochloric acid.

Numerous lipid-purification methods using organic solvent extraction have been developed, as well as several nonsolvent methods. Among the former, the choice of solvent depends on the charge character of the lipid being isolated:

  • Polar lipids (such as phospholipids) are most soluble in alcohols and other polar solvents
  • Nonpolar lipids (such as triacylglycerols) are most soluble in nonpolar solvents such as hexane and pentane

Nonsolvent liquid extraction methods typically involve treatment with sulfuric acid, or chemical surfactants in situations where corrosive acids are to be avoided.

Further separation of purified lipids into subtypes, and performing other analytical operations, are accomplished through one or more instrumental and/or chemical methods and techniques including:

  • Thin-layer chromatography (TLC)
  • Gas chromatography (GC)
  • Mass spectrometry (MS)
  • Nuclear magnetic resonance (NMR)
  • Iodine value method (measuring a lipid’s iodine-absorbing capacity)
  • Saponification number method (quantitating KOH needed to break down a lipid into its glycerol and fatty acid components)

Newer lipid purification methodology plays a central role in the burgeoning field of extracellular vesicle (EV) research. Similar to the membranes of cells from which they’re derived, EVs involved in intercellular communication are themselves delimited by phospholipid bilayers or monolayers.

Conventional protocols for purifying EVs using precipitation and ultracentrifugation have been replaced in some cases by affinity-based methods. For example, EVs such as exosomes and microvesicles express phosphatidylserine on their surfaces, which binds specifically to Tim4 proteins that can then be selectively isolated, resulting in EVs of significantly greater purity than can be achieved through traditional methods.

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