Lipid Aggregation in Water

The intent of this exercise is to demonstrate the behavior of various lipids when they are forced to disperse in water. Evident differences in their behavior are related to their molecular structures and resulting differences in physical properties and modes of aggregation. Lipids used are triglyceride, lecithin, and cholesterol.

I provide 10% (w/v) solutions in ethanol/ethyl ether (1:1, v/v) of triglyceride (vegetable oil), lecithin (see below), and cholesterol and a fourth solution that is 6% triglyceride, 3% lecithin, and 1% cholesterol. Both solutions containing triglyceride are dyed red with oil red O to improve later visibility of triglyceride in their dispersions. All solutions are homogeneous (though sometimes they separate when stored cold). Each solution has a glass 1 mL tuberculin syringe with a (filed-off) 18 G needle attached.

With the tip of the needle about a cm above the water, students rapidly squirt 0.5 mL of each solution into separate 18 x 150 mm test tubes containing 10 mL of water, then stopper and invert the tubes quickly. As the solvent quickly disperses into the water, the uniformly dissolved lipid molecules aggregate in a manner dictated by the balance of favorable and unfavorable interactions with water, with visible results. I have them incubate their tubes in a 60 degree water bath for thirty minutes to drive off at least some solvent, further force the entropically-driven lipid interactions, and use up a little time.

Triglyceride separates into floating oil droplets, which are distinguishable from air bubbles by their red color. Lecithin gives a slightly hazy, but otherwise stable dispersion. Cholesterol separates into a solid, chunky precipitate that either sinks or floats depending on how much air it has entrained. Most impressively to me, the lipid mixture gives a decidedly milky dispersion (pink in color) which, like the lecithin, is otherwise stable with time. The droplets can in fact be seen with a microscope, though I haven't yet had the students examine them; I haven't looked to see whether anything is visible with lecithin (which, according to lore, forms small single-walled vesicles with diameters on the order of dozens to hundreds of nanometers and wouldn't be expected to be visible with a light microscope).

Students have been primed to molecular events by an introduction that discusses how lipids' molecular structures influence their interactions with water. At the end of the exercise, I ask them (1) to describe what they observe before and after the 30-minute incubation, (2) to explain how their observations correlate with each lipid's aggregation properties, and (3) to draw a cartoon of the organization of the mixed lipid droplets in their fourth tube.

Crude Lecithin. Pure lecithin (phosphatidylcholine) is very costly in the amounts used here. Sigma sells an inexpensive crude soy lecithin preparation that is about 15% lecithin, but isn't (fully) soluble in ethanol/ether. To prepare a more enriched fraction of lecithin, I dissolve a gob of the crude soy stuff in hot ethanol, let it cool, separate the solution from the sticky insoluble goo, and dry it to near constant weight with a boiling water bath and air stream. The result is another yellow-brown sticky goo that is readily soluble in ethanol/ether and which I use in the exercise.

I have played around briefly with solvents other than ethanol/ether. There aren't many that both (a) dissolve both triglyceride and lecithin and (b) are water-miscible. Dioxane, for example, fits these criteria but gave less satisfactory dispersions.