Isolation of Cholesterol
Preparation: Mayo, et. al. pp. 129-135. Look up and record the melting point of pure cholesterol here. Cholesterol, the steroid in highest concentration in the body, is the substance from which other naturally occurring steroids are biosynthesized. Further details on cholesterol are given in McM p.139 and 1117.
Procedure: For a continuous extraction of gallstones,
set a hot plate to 75±5 oC; normally this will
require a setting of 60% of maximum (note 1). While this
temperature is adjusting, introduce 1 mL of 2-butanone to a 50 mL
beaker and mark the beaker. Then add another 3 mL of 2-butanone
and a small (1 mm) boiling stone with a forceps. Next, trim a
paper cone formed from 9 cm diameter disk of filter paper (note
2) to fit snugly, point down, resting on the flare of the beaker.
Several trials may be necessary for a proper fit; before placing
the cone for the last time in the beaker, cut a 1/2 cm notch at
the cone's edge to allow vapors to pass around this cone.
Carefully load 100 mg of autoclaved and ground gallstones (note
3) into the filter cone and then heat the beaker to boil the
2-butanone. Cover the beaker with a watch glass and place a 30 mL
beaker filled with ice on top of the watch glass; this will serve
to condense the butanone.
Allow 30 minutes for the extraction. You should note the
vapors condensing under the bottom of the watch glass, then
dropping into the filter cone containing the ground gallstones
and then returning to the boiling solution. Make a careful
drawing of this apparatus in your notebook.
In order to purify the crude cholesterol by recrystallization,
remove the watch glass and the filter cone and allow the solution
to concentrate to the 1 mL mark. Add 1/2 mL of methanol and while
the solution is still boiling, add water dropwise (pipet) until a
white or off-white cloudiness persists. If too much water is
added, add just enough butanone to redissolve the solid while
hot. Then set the saturated solution to cool in ice. Collect the
solid using a Hirsch funnel fitted with a paper disk; apply
suction by fitting the funnel to a filter tube connected to
aspiration. Rinse the beaker 2-3 times with 1/2 mL of water.
Press the white crystals and maintain the suction until the
crystals are dry. Determine the melting point of the crude
product. Scrape the product into a preweighed plastic bag then
reweigh and seal the bag and attach it to an empty page in your
notebook. Report the amount of cholesterol obtained. Average
yield and melting point: 70 mg, 144-5o C.
Notes: 1.Careful control of the temperature is
important; the solvent should be just hot enough to boil.
Overheating will cause the solvent to evaporate excessively.
2. The filter should not be pleated. It is unlikely that the
filter cone will dislodge from its position, but if it does,
remove the assembly from the hot plate, remove the watch glass
and straighten the filter cone using a forceps. From time to time
add more solvent if its level is below the 1 mL mark; also
replace the water in the watch glass with fresh ice.
3. Gallstones are obtained from hospitals and have the
appearance of round stones. They are autoclaved then crushed
between pieces of paper with a hammer to a fine powder. The paper
effectively absorbs any moisture in the gallstones. The material
to be extracted should be in powder form.
References:
Vestling, M.M. Journal of Chemical Education, 1990, 67, 274.
Williamson, K.L. Macroscale and Microscale Organic Experiments, 2nd ed., Heath: Lexington, MA, 1987; p. 153
"Mayo et al.": Mayo, D.W., Pike, R.M., Butcher, S.S. and Trumper, P.K. Microscale Techniques for the Organic Laboratory; Wiley: New York, 1991
"McM": McMurry, J. Organic Chemistry, 4th ed., Brooks/Cole Publishing Company, Pacific Grove, CA. 1996
Rev. December, 1998