How to Isolate Cholesterol from Gallstones

Step-by-Step Guide

1. 1

   Step 1: Obtain gallstones.

   In some places, you may request gallstones from the department of surgery or the department of pathology of hospitals.
2. 2

   Step 2: Place the gallstones between sheets of paper

   ,, Record the weight.
   
   This will be important to estimate how much solvent to use. ,, Stir gently.
   
   The solution will have a dirty-yellow to brown appearance from the bile pigment bilirubin. , 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. ,, 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. , Wrap cotton padding or toweling around the hot container for insulation to allow slow cooling, then let it cool undisturbed to room temperature. ,, 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.
3. 3

   Step 3: then crush them by pounding lightly with a hammer.

4. 4

   Step 4: Sterilize the gallstones by washing them with household bleach (5% sodium hypochlorite solution).

5. 5

   Step 5: Weigh out the crushed gallstones.

6. 6

   Step 6: Add the crushed gallstones to a reaction container.

7. 7

   Step 7: Add 2-butanone (methyl ethyl ketone)

8. 8

   Step 8: about 10 cc per gram of crushed gallstones

9. 9

   Step 9: to the reaction container and heat to a gentle boil.

10. 10

    Step 10: Filter the hot solution through a paper filter disk/cone on a funnel into another clean container.

11. 11

    Step 11: Add methanol (caution: extremely toxic "wood alcohol") to the filtrate

12. 12

    Step 12: about 5 cc per gram of starting crushed gallstone.

13. 13

    Step 13: Reheat the filtrate to a gentle boil

14. 14

    Step 14: then add water drop by drop until a very faint cloudiness appears.

15. 15

    Step 15: Carefully remove the solution from heat.

16. 16

    Step 16: Once at room temperature

17. 17

    Step 17: unwrap and cool the container in ice to allow more cholesterol to crystallize out of solution.

18. 18

    Step 18: Collect the cholesterol by filtration.

Detailed Guide

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