Analysis of Lipids in Egg Yolk and Milk

In this exercise, students extract lipids from milk and egg yolks and separate the mixture by thin-layer chromatography. They review the different lipid classes found in animal fat and consider how their molecular structures influence the separation techniques used.

I make a mixture of egg yolk diluted with 5 volumes of 1 M NaCl. Whole milk is made 1 M in NaCl. Students mix 1 mL of dilute egg yolk or of milk with 1.5 mL of isopropyl alcohol in a small test tube. (If you have them, screw-capped tubes with Teflon-lined caps are superior, but corks are fine.) They then add 1 mL of petroleum ether (which they are warned is very flammable), mix well, and allow them to stand for a minute or so. After removing the upper layer to a clean test tube, they remove 10 microliters to spot on a 5 x 10 cm TLC plate on which they also spot 10 microliters of 0.2% solutions of lecithin, cholesterol, and triglyceride (I use olive oil) in chloroform (other solvents should work fine). I use Merck plates, silica gel 60; the organic binder is very hard and is easily marked with pencil.

Plates are developed in a 400-mL beaker covered with aluminum foil containing 10 mL of developing solvent (petroleum ether/ethyl ether/acetic acid in a volume ratio of about 75:25:1). It takes about 15 minutes. After drying in the hood, the plates are wet, using a sprayer or a dropper, with 10% ammonium sulfate in water, blotted dry, and placed on a hotplate to char. This can be a little tricky. As long as you don't heat too hot, only the lipid spots char; too hot and the background chars (or the plate cracks). If you don't heat hot enough, spots char too slowly (or not at all). You may want to play with your hotplate a bit to find what works best. (I tried detecting spots with iodine vapors for several years, but cholesterol stains very poorly with iodine.)

The results are pretty clear. Lecithin doesn't move at all off the origin, cholesterol moves about halfway up the plate, and triglyceride moves near the solvent front. They seem to be the only three spots in the yolk extract; there is a suggestion of some other spots in the milk.

Finally, students are asked (1) which lipids are evident in their extracts, (2) which seem most/least abundant, (3) whether there appeared to be any other lipids than the standards, (4) to explain the relative migration of the three standards from their structures and the relative polarities of the solvent mixture and the plate material, and (5) why proteins aren't soluble in the solvent mixture.