Lipid Chemistry
There are four parts to this exercise: observation of lipids' physical properties, estimation of unsaturation,
and qualitative analyses for esters and cholesterol.
In the first part, students are shown authentic samples of several lipids: pure fatty acids (six- to eighteen-carbon saturated, oleic, and linoleic), triglycerides (canola, coconut, olive, and soybean oils, lard, and shortening), and cholesterol. For the fatty acids, they are asked to complete a data table in which they describe physical properties (state and color) of all fatty acid samples and to draw the complete structures of stearic, oleic, and linoleic acids. For the triglycerides, they are to complete a data table recording physical properties and indicating,
based on physical state, whether saturated or unsaturated fatty acids are more abundant in each. For cholesterol, they are to describe its physical properties and draw its structure.
For the next three parts, students are provided with 5% solutions of stearic acid, lard, coconut oil, olive oil,
shortening, and soybean oil, and a 0.05% solution of cholesterol,
all in methylene chloride.
To determine degree of unsaturation, students put fifteen drops of solutions of the first
six lipids above into dry test tubes. To these, they add bromine
solution dropwise until the color of the bromine persists and record the number of drops they used. Bromine solution is prepared by dissolving 4 mL bromine in 250 mL methylene chloride. Students are cautioned about the tendency of methylene chloride solutions to
drip or spurt from droppers.
The presence of esters is determined by the classic ferric hydroxamate test.
Students put two drops of lipid solution into dry test tubes; minimally I have
them test stearic acid, coconut oil, and cholesterol. To these, they add 2 mL of
a 3:1 mixture of 95% ethanol and 1-butanol (it's important not to use denatured
ethanol, which usually contains some ethyl acetate or other ester). Next they
add two drops each of 2 M hydroxylamine hydrochloride and 3 M NaOH, mix well,
and let the tubes stand for at least five minutes. Finally, they add two drops
each of 6 M HCl and 2% ferric chloride hexahydrate (in 0.1 M HCl) and mix well.
Samples with esters will give a burgundy color against the yellow background of
the ferric chloride. For the Liebermann-Burchard test for cholesterol, students put 10 drops of the above solutions of cholesterol, coconut oil, or lard in separate dry test tubes and add 3 drops of acetic anhydride and one drop of concentrated sulfuric acid. Formation of a green or green-blue color after a few minutes is positive.
Ideally, the cholesterol solution gives a nice reaction, the coconut oil should show no significant color
change, and the lard gives a weak reaction. The results for this test have been variable over the past few years.
For example, coconut oil sometimes gives a green color, and unsaturated oils
invariably do, whereas lard often gives a brown color. I think the
variability has been due to using old solutions; they should be made fresh
with reasonably fresh samples, which is fairly inconvenient.
The exercise wraps up with a set of questions about the molecular basis of
different physical properties of fatty acids and the differences in degree of
unsaturation between shortening and soybean oil (from which it is made), and asking them to interpret their results in terms of popular generalizations about the animal or vegetable origins of cholesterol and unsaturated and saturated triglycerides.
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