Cholesterol Lab Part 1
How to Isolate Cholesterol from Gallstones
Cholesterol is a natural organic steroid and alcohol compound found in cell membranes, where it is an essential structural component of mammalian cell membranes and functions to establish proper membrane permeability and fluidity. In addition, cholesterol is an important building block for synthesis of bile acids, steroid hormones, and several fat-soluble vitamins and is found in the spinal cord, brain, and nerve tissue.
The gallbladder concentrates bile produced from the liver and releases them as needed for digestion. The gallbladder also stores free cholesterol, which can precipitate to form gallstones if its concentration in the bile exceeds a critical level. Accumulation of gallstones can irritate the lining of the gallbladder, causing pain, nausea, and vomiting. They may also block bile ducts and cause complications requiring surgery (cholecystectomy) to remove the gallbladder with its gallstones. The following steps will help guide you to isolate cholesterol from gallstones.
Obtain gallstones.In some places, you may request gallstones from the department of surgery or the department of pathology of hospitals.
Place the gallstones between sheets of paper, then crush them by pounding lightly with a hammer.
Sterilize the gallstones by washing them with household bleach (5% sodium hypochlorite solution).
Weigh out the crushed gallstones.Record the weight. This will be important to estimate how much solvent to use.
Add the crushed gallstones to a reaction container.
Add 2-butanone (methyl ethyl ketone), about 10 cc per gram of crushed gallstones, to the reaction container and heat to a gentle boil.Stir gently. The solution will have a dirty-yellow to brown appearance from the bile pigment bilirubin.
Filter the hot solution through a paper filter disk/cone on a funnel into another clean container.Apply a gentle vacuum in the receptor container to facilitate filtration. Rinse the cooking container with a small amount of hot 2-butanone and transfer that rinse to the funnel. The cholesterol will be completely dissolved in the 2-butanone solvent, while insoluble impurities (mostly composed of bilirubin, a metabolite of hemoglobin, that gives the gallstones a brown color) will be filtered out during this extraction process.
- If the filtrate remains highly coloured, add activated charcoal and re-filter.
Add methanol (caution:extremely toxic "wood alcohol") to the filtrate, about 5 cc per gram of starting crushed gallstone.
Reheat the filtrate to a gentle boil, then add water drop by drop until a very faint cloudiness appears.The cholesterol is very soluble 2-butanone, less soluble in methanol, and insoluble in water. Adding the water as a precipitant into the solution will help saturate the solution with cholesterol for crystallization. Methanol is needed to bridge the solvents, as 2-butanone and water are not very miscible with each other, and methanol is miscible with both water and 2-butanone.
Carefully remove the solution from heat.Wrap cotton padding or toweling around the hot container for insulation to allow slow cooling, then let it cool undisturbed to room temperature.
Once at room temperature, unwrap and cool the container in ice to allow more cholesterol to crystallize out of solution.
Collect the cholesterol by filtration.The yield is large colorless crystalline plates and needles of cholesterol, about 75% of starting crushed gallstones. Save the filtrate. If greater yield is desired, you can obtain a second crop of less purity by boiling the filtrate, adding water drop by drop, and repeating the crystallization process.
- It is best to carry out this procedure in a controlled, well-ventilated area.
- High cholesterol in a high ratio of LDL (bad) to HDL (good) cholesterol leads to the deposition of cholesterol both in the gall bladder (resulting in gallstones) and on the walls of the arteries (as a plaque restricting blood flow and causing hardening of the arteries, ie: atherosclerosis).
- Both methanol and 2-butanone are highly volatile and flammable.
- Methanol is extremely toxic and causes blindness if imbibed.
- Butanone is an irritant, but serious health effects in animals have been seen only at very high levels. When inhaled, these effects included birth defects.
- Butanone is listed as a Table II precursor of chemicals frequently used in the illicit manufacture of narcotic drugs and psychotropic substances under International Control, International Narcotics Control Board.
Things You'll Need
A hot plate
Filtration apparatus (funnel, paper filter disk/cone, clean container to catch the filtrate, and vacuum suction)
Sources and Citations
- Mayo, D.W., Pike, R.M., Butcher, S.S. and Trumper, P.K. Microscale Techniques for the Organic Laboratory; Wiley: New York, 1991
- Schwetz et al. (1991). "Developmental toxicity of inhaled methyl ethyl ketone in Swiss mice". Fund. Appl. Toxicol. 16: 742–748.
Video: Isolation of Cholesterol
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