Experiment #2 - Part 2
Adam Capriola
10/17/07
Dr. Murray
“Natural Products Isolation – Cholesterol from Gallstones”
Lab Partner: David Lemon
I. Introduction
A. Objective
The purpose of this experiment is to isolate cholesterol from gallstones via the techniques
of extraction and recrystallization. The gallstones will first be dissolved in 2-butanone heated in
a hot sand bath. The solution will then be transferred to a micro column using a pipet to filter
out the bilirubin, which is the primary impurity of gallstones. The remaining 2-butanone will be
removed and the crude cholesterol left over will be recrystallized by dissolving it with methanol
and centrifuging it in a Craig tube.
B. Materials and Safety
Chemical
Name
Molecular
Formula
Molecular
Weight
(g/mol)
72.11
Liquid
Solid
Solubility
Potential
Hazards
b.p.
ºC
80
Density m.p. ºC
g/mL
0.8050 −86
H2O
2-Butanone
C4H8O
Cholesterol
Bilirubin
Anhydrous
magnesium
sulfate
Anhydrous
sodium
sulfate
Methanol
C27H46O
386.66
C33H36N4O6 584.662
MgSO4
120.369
360
---
1.067
<1
2.66
149-150
192
1124
H2O
Insoluble
H2O
Flammable,
Irritant
Irritant
Irritant
n/a
Na2SO4
142.04
--
--
241
H2O
Irritant
CH3OH
32.04
64.7 0.7918
–97
H2O
Flammable,
Toxic
C. Experimental Procedure
A sample of crushed gallstones weighing about 100 mg will be obtained and
weighed. The crushed gallstones will then be placed in a 10 x 100 nm reaction tube along with a
boiling stick and 1.5 mL of 2-butanone. This mixture will be gently heated in a hot sand bath.
In a second reaction tube also containing a boiling stick, 1.0 mL of 2-butanone will be heated
until it is boiling. When the gallstones have disintegrated and the cholesterol has dissolved in the
first reaction tube, it will be filtered through a micro column. The micro column will be
prepared from a Pasteur pipet packed with a loose wad of cotton, 2 mm of sand, 3-4 mm of
anhydrous magnesium sulfate, 1 cm of anhydrous sodium sulfate, 1 cm of Norit RO 0.8 activated
carbon pellets, and a very small piece of cotton. The micro column will be clamped in a vertical
position and will have a weighed and cleaned 10 x 100 mm reaction tube beneath it.
The solution in the original reaction tube will be transferred into the micro column using
a warm Pasteur pipet, which is warmed by immersing it in the hot vapors of the boiling 2butanone. The 1.0 mL of boiling 2-butanone will be used to wash out the original reaction tube
and pipet into the micro column. The filtered hot solution of 2-butanone solution will then be
warmed in a sand bath and the 2-butanone will be removed under a gentle stream of nitrogen.
The crude cholesterol will be scraped from the reaction tube onto a previously tared piece of a
creased, glazed weighing paper. After obtaining the weight of the crude cholesterol, it will be
placed into a Craig tube. To recrystallize the cholesterol, it will be first dissolved in the
minimum amount of hot methanol. The solution will then be allowed to slowly cool to room
temperature, and it will then be cooled in an ice bath. The crystalline cholesterol will be isolated
via centrifugation. The cholesterol will then be allowed to air dry. It will then be weighed and
its melting point will be determined.
II. Experiment and Results
A. Data
First, a sample of crushed gallstones weighing 0.131 g was obtained. Next, a micro
column was prepared using a Pasteur pipet packed with a loose wad of cotton, about 2 mm of
sand, about 3 to 4 mm of anhydrous magnesium sulfate, about 1 cm of anhydrous sodium sulfate,
about 1 cm of Norit RO 0.8 activated carbon pellets, and a small piece of cotton. One prepared,
the micro column was clamped vertically on a ring stand. The crushed gallstones were then
placed in a clean glass centrifuge tube along with a boiling stick and 1.5 mL of 2-butanone. The
solution was gently heated in a sand bath, along with a separate centrifuge tube containing about
1.0 mL of 2-butanone. One the gallstones dissolved, the solution was transferred to the micro
column using a warm pipet. The pipet was warmed by immersing it into the hot vapors of the
second tube containing boiling 2-butanone. The reaction tube was rinsed with the hot 2butanone and the remaining solution was filtered in the micro column.
The filtrate was collected in a clean centrifuge tube. The 2-butanone was removed from
this tube under a stream of nitrogen and the remaining cholesterol was allowed to air dry for a
week. A minimum amount of methanol was added to the cholesterol and the tube was heated in
a sand bath along with a boiling stick until the cholesterol dissolved. The solution was then
allowed to cool to room temperature and was then put into an ice bath to cool more. The
crystalline cholesterol formed was isolated via centrifugation. The crystals were removed from
the tube using a spatula onto filter paper and the crystals were allowed to air dry for a week.
Weight of Gallstones (g)
Weight of Crystalline Cholesterol (g)
Melting Point of Crystalline Cholesterol (ºC)
0.131
III. Conclusions
Without knowing my final weight and melting point of the crystalline cholesterol, I can
only discuss possible sources of error during the procedure. When preparing the micro column,
if the wrong amount of any of the materials was added, the bilirubin may not have filtered out.
This make the amount of cholesterol recovered be less than expected because the cholesterol
may not have crystallized if the bilirubin was still in the solution. If the filtered solution was
cooled too fast, that may have prevented the formation of crystalline cholesterol, too.