In this exercise, students gain familiarity with three separation methods used in lipid biochemistry: extraction and column and thin-layer chromatography. The general approach is to extract total lipids from egg yolk using methanol and chloroform, then separate the lipid fractions (triglyceride, cholesterol, and phospholipids) by chromatography on silica gel. The fractions are analyzed by thin-layer chromatography here and further in the next exercise.

Extraction of Lipids: I prepare ahead of time a dispersion of egg yolk lipids in 1 M NaCl (see below). Students mix 1.6 mL of this dispersion with 6 mL of methanol/chloroform (2:1 by volume) in a test tube (16 x 125 mm) with a Teflon-lined screw-cap. After shaking the tube, they add 2 mL each of 1 M NaCl and chloroform. Again the tube is capped and shaken, then centrifuged briefly to separate the phases. After removing and discarding the upper phase, they remove the lower phase to a clean preweighed tube and apply five microliters to two TLC plates (below). Solvent is removed from the tube by drying under a stream of nitrogen or air in a warm water bath. When the residue is dry the tube is weighed. The residue is the total lipid pool from the yolk and can be stored cold if necessary.

Column Chromatography: To separate the various lipid fractions, students prepare a small column by pouring a slurry of 0.5 g of silica gel in 4 mL of petroleum ether into a glass column with a tapered end plugged with glass wool. They dissolve the lipid residue in 2 mL of petroleum ether and pour it into the column, saving the run-through, which contains some triglyceride, in a preweighed tube. To complete elution of triglyceride, they wash the column with 9 mL of a 9:1 mixture of petroleum ether and ethyl ether, collecting the eluate in the same tube as the run-through. Cholesterol is then eluted into a clean preweighed tube using 9 mL of 5% methanol in chloroform. Finally, to completely remove phospholipids, students elute the column into a screw-capped tube with 9 mL of chloroform/methanol/water (1:3:1 by volume). To this tube they then add 2 mL of chloroform and 2 mL of 1 M NaCl to break phases and get rid of the water; the lower phase is removed to a clean preweighed tube.  Ten microliters of the triglyceride and cholesterol eluates and five microliters of the phospholipids are applied to their TLC plates. Solvent is removed from the tubes again as above and the tubes are weighed. Fractions are saved for qualitative analysis in the next lab period (next exercise).

Thin-layer Chromatography: Students are given two 4 x 10 cm TLC plates (Merck Silica Gel 60). After applying samples as described above, they develop the plates in 400-mL beakers covered with aluminum foil containing 10 mL of developing solvent mixtures. One solvent mixture is petroleum ether/ethyl ether/acetic acid (75:25:1 by volume) and the other is chloroform/methanol/acetic acid (65:25:10 by volume). Spots are located by spraying the plates with 20% ammonium sulfate, blotting them, and heating for a few minutes on a hot plate. Although these plates have an organic binder that might interfere with this charring procedure, it hasn't been a problem for me yet.

For a brief write-up, students are asked to summarize their recovered masses at each step, draw a sketch of their TLC results, and comment on the apparent purity of their various fractions.

Preparation of Egg Yolk Lipid Dispersion: I dilute an egg yolk with three volumes of 1 M NaCl, mix well, and reserve about 3 mL. The rest is extracted with an equal volume of ethyl ether. It gives an outrageous emulsion that takes some time to separate by centrifugation, but eventually it gives two layers. Ether is removed by rotary evaporation and lipids are dispersed in the same volume of 1 M NaCl as was extracted and mixed with the small volume of the original dilution.  That may help stabilize the dispersion and gives them some protein in their extractions, but much less than the mess they would have if they extracted diluted egg yolk directly. This dispersion seems to be stable for at least a year in the freezer.

Revised 6/13/07