Isolation of Cholesterol from Egg Yolk To the Instructor Cholesterol is nicely crystalline and is easy to handle.

The student can easily bring a hard-boiled egg yolk to lab. We ran this experiment through twelve sections of first semester sophomore organic students without difficulty, with half taking each option. The two options take about the same elapsed time. There is a direct tie-in with human health in this lab, because of the problem of excess cholesterol in the diet. The TLC experiment, following the oxidation and then conjugation, illustrates enolization and double bond equilibration. It can be used either qualitatively, or quantitatively, with isolation by column chromatography of the product cholestenone. The purification of the cholesterol with oxalic acid is an intriguing example of specific complex formation. Comparing the 13C NMR of cholesterol 1 to the 13C NMR cholestenone 3 teaches a useful lesson about chemical shifts of normal vs. polarized alkenes. We use glass-backed TLC plates, visualized by dipping in ceric ammonium molybdate solution and heating on a hotplate in the hood. The usual iodine chamber also works. Note that the 13C NMR spectra are included were recorded using a JVert pulse sequence, so that methylenes and quaternary carbons are up, and methyl groups and methines are down. This will help the curious student begin to make assignments, even with such complcx structures. Sample Pre-Lab Quiz: 1. Sketch a mixture of ether and water in a separatory funnel. Label the layers. Which one would contain cholesterol? 2. Draw the structure of oxalic acid. 3. Draw the mechanism for the reaction of cholesteryl acetate with methanol and methoxide. Cholesterol is one of the products of the reaction. What is the other product? 4. Draw the mechanism of chromic acid oxidation of a secondary alcohol to the ketone [They should know this one the instructor can then draw the analogy to hypervalent iodine].

Supplies: For 12 students, assuming half do each option: Flash silica gel (40-60 ) 250 g

Sand 50 g K2CO3 12 g TLC plates 90 EtOAc 700 mL Pet Ether 3.5 L Methanol 120 mL Cyclohexane 240 mL 1,2-Dichloroethane 100 mL CH2Cl2 400 mL Acetone 100 mL Oxalic acid 5 g Dess-Martin reagent 6 mL of 3M in CH2Cl2 (Aldrich) pTsOH 5 g