Students ideally will have separated triglyceride, cholesterol, and phospholipids from egg yolk in the previous exercise and will use three assay methods to identify lipids present in each of the fractions by testing for carboxylate ester, phosphate, and cholesterol.

Students will know the mass of lipids they recovered in each fraction and will add sufficient chloroform to make 10 mg/mL solutions. For knowns, I give them chloroform solutions of olive oil (1%), cholesterol (0.5%), and phosphatidylcholine (dioleoyl, 2%). The exercise is quite short and the results should be clear. The only (small) catch was for students to remember that phospholipids contain ester as well as phosphate.

Ester assay: Students place two drops of sample in a test tube and add 0.5 mL of ethanol/1-butanol (3:1 by volume; don't use denatured ethanol which typically contains esters).  They then add sequentially two drops each of 2 M hydroxylamine hydrochloride and of 3 M NaOH and mix well. After allowing the samples to stand for 5 minutes, they add two drops of 6 M HCl and one drop of 5% ferric chloride (hexahydrate) in 0.1 M HCl and mix well. Carboxylate esters are converted to hydroxamic acids which form a magenta-colored complex with ferric ion.

Phosphate assay: Students put two drops of sample into a clean test tube and add 0.5 mL of 10% magnesium nitrate (hexahydrate) in 95% ethanol. In a hood (noxious nitrogen dioxide is formed) the tube is heated over a flame until the solvent evaporates and the magnesium nitrate is decomposed. When a white residue forms and brown gas stops evolving, heating is stopped and the tube is allowed to cool. This step destroys organic parts of the phospholipid structure and forms magnesium pyrophosphate from any organic (or inorganic) phosphate present in the sample.

Students then add 0.5 mL of 2 M HCl and mix to dissolve the solid residue, then heat in a boiling water bath for 5 minutes to hydrolyze pyrophosphate to phosphate. They finally add 0.5 mL of 6% ammonium molybdate in 4 M nitric acid. Formation of a yellow color, which may be followed by slow formation of a fine yellow precipitate, indicates the presence of phosphate.

Cholesterol assay: This is essentially the same assay (the Liebermann-Burchard test) as I've described for another course, CHM 1102. Students place two drops of sample in a test tube and add 0.25 mL of chloroform.  They then add six drops of acetic anhydride and two drops of concentrated sulfuric acid and mix well. A greenish color produced after a few minutes is indicative of cholesterol. Results are quite sensitive to the presence of water, so keep everything dry. Supposedly this test is also quantitative, with maximal absorbance near 680 nm. I have no idea how specific this is for cholesterol (compared with, say, various plant sterols), and no one seems to know what the chemistry of the chromophore is.

For their write-up, students were asked to describe their results and conclusions about what lipid(s) each fraction contained. This exercise could be made more quantitative. There are relatively simple quantitative ester and phosphate assays which students could use to determine, for example, average molecular weights and ester:phosphate ratios.

Revised 6/13/07