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Purification of Cholesterol
An Oxidative Addition-Reductive Elimination Sequence
From your lectures sessions in CHEM 2010 you have learned that elimination
reactions may occur when alkyl halides are treated with strong base. With strong base,
the mechanism is generally E2, as shown in the figure below.
-
OH
H
Br
The orbitals of the atoms involved in the bond breaking sequence must be aligned
in an ‘antiperiplanar’ arrangement to provide maximum orbital overlap in the transition
state. This reaction is neither an oxidation nor reduction according to the convention of
electron counting, since both an electropositive atom (H) and electronegative atom (Br)
are lost.
Another important type of elimination reaction is known as reductive elimination.
In this reaction, a reducing agent (often powdered Zn metal) is used as the electron
source. While the exact mechanism is not fully known, it can be envisioned
mechanistically as shown in the following figure.
Zn
Br
+
Br
ZnBr
+
+
Br
-
ZnBr2
This reaction is considered an oxidation-reduction reaction, since the Zn metal is
oxidized to Zn+2 and the dibromocyclohexane is reduced (two electronegative Br atoms
are lost).
In this experiment, you will use a reductive elimination reaction in the
purification of crude cholesterol. Cholesterol is a steroidal compound present in animal
cells. In spite of its bad reputation, cholesterol is vital for life. It is a structural
component in cell walls and is also required for the formation the myelin sheath covering
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nerve bundles. It is also a precursor for other important steroidal hormones. Cholesterol
isolated from natural sources is generally contaminated with ~3-5% related compounds.
The structure of cholesterol and related impurities is shown in the following figure.
H3C
H3C
CH3
CH3
H
CH3
CH3
H
CH3
H
H
HO
H
CH3
H
CH3
H
CH3
H
HO
3β−cholestanol
cholesterol
H3C
CH3
H3C
CH3
H
CH3
CH3
CH3
CH3
H
CH3
CH3
H
H
H
H
HO
HO
5,7-cholestadien-3β−ol
7-cholesten-3β−ol
Because of steric constraints imposed by the steroid ring structure, only cholesterol reacts
with bromine to form an insoluble diaxial dibromo compound, as shown below.
Cholestanol does not react with bromine, whereas the two remaining impurities are
dehydrogenated by bromine to form soluble dienes and trienes by a mechanism we will
not consider here.
H3C
CH3
H
CH3
CH3
CH3
H
CH3
H
CH3
H
CH3
Br 2
H
HO
H3C
CH3
H
H
Br
HOAc
HO
H
Br
The dibromocompound has limited solubility in the reaction solvent and precipitates as a
solid. It can be purified by solvent washing or crystallization techniques and separated
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from the impurities which remain soluble in the reaction solvent. Once the dibromo
compound has been purified, it can be reacted with zinc to regenerate cholesterol free of
the impurities.
H3C
CH3
H3C
CH3
H
CH3
H
CH3
CH3
H
CH3
H
CH3
CH3
Zn
H
Br
H
H
MTBE
H
HO
HO
Br
________________________________________________________________________
!
"
%
# $
"
"
"
&
' (
Reagent
cholesterol
MTBE
bromine
acetic acid
sodium acetate
zinc
10% NaOH
methanol
formula
C27H46O
NaOH
MW
mp
bp
Density
na
Comments
flammable
toxic, corrosive
40
na
Updated 6/20/2005
na
na
na
1.11
Corrosive
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Procedure:
Note: Ethers are common solvents used in many organic synthesis preparations. They
have the general formula R-O-R’. Ethers dissolve many organic substrates, yet are
unreactive with many reagents; these characteristics make them useful solvents. A
common ether solvent is diethyl ether. It is commonly referred to simply as ‘ether’ and
was often used as a general anesthetic prior to the development of improved substitutes.
Its low boiling point (35oC) and its propensity to form explosive peroxides on standing
are safety concerns. This procedure uses t-butyl methyl ether as the solvent. It has a
higher boiling point than diethyl ether and is less prone to form unstable peroxides. This
compound is generally known as MTBE and was added to certain gasoline blends to
provide more complete combustion. However it dissolves in water to the extent of 4.8g
MBTE per 100g water. Concerns about groundwater contamination from leaking
gasoline storage tanks have led to a phase out of this use.
A: Bromination of cholesterol
1. In a 50mL Erlenmeyer flask, dissolve approximately 1g (recorded to three decimal
places) of crude cholesterol in 7mL t-butyl methyl ether (MTBE) by gentle warming
on a hot plate (setting 4-5).
2. Using a graduated pipet, add 5mL of the bromine/sodium acetate reagent (this has
been prepared from 9.0g bromine, 100mL acetic acid and 0.8g sodium acetate) to
the Erlenmeyer flask in step 1. Record your observations over several minutes
3. After the reaction appears to stop, place the flask in an ice bath while stirring with a
glass rod for approximately 10 minutes.
4. Collect the solids by Buchner filtration and wash with a cold solution prepared from
3mL MTBE and 7mL acetic acid. to remove the entrained mother liquors from the
solid.
5. Wash the solids with cold methanol and suck dry for about 5 minutes.
6. Transfer the solids to a pre-weighed 50mL Erlenmeyer flask and record the weight of
the dibromo compound.
B: Reductive Elimination
7. Add 20mL MTBE to the dibromo compound in step 6 followed by 5mL of acetic
acid. Swirl the contents to mix the solvents.
8. Add 0.2g Zn dust and continue swirling. Record your observations.
9. After swirling for 5-10 minutes, add water dropwise until any solids (other than the
zinc) dissolve (no more than 0.5mL water should be necessary)
10. Decant the solution from the zinc into a small separatory funnel and add 20mL water.
Shake and drain the water layer into a 150mL beaker. Add an additional 20mL water
and repeat.
11. Add 20mL 10% NaOH solution to the separatory funnel, shake, settle, and drain the
aqueous layer into the same 150mL beaker in step 10.
12. Add 20 mL saturated sodium chloride solution to the MTBE layer in the separatory
funnel, shake, settle and drain. (The use of saturated salt solutions will lower the
water content of the MTBE layer by drawing the water into the highly ionic salt
solution-a process similar to osmosis)
13. Dry the MTBE layer with a small amount of magnesium sulfate; filter through a
Chem-wipe tissue into a 50mL beaker to remove the hydrated magnesium sulfate.
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14. Add 10mL methanol and a boiling stone to the MTBE solution. Evaporate the
solvent gently on a hot plate (use the snorkel hood!) until solids begin to form or the
volume of the solution reaches ~5-10mL.
15. Remove the beaker from the hot plate and allow the contents to cool to room
temperature. If no crystals have formed, scratch the surface of the beaker with a glass
rod. Place the beaker (carefully-no spills now) into an ice bath for about 15 minutes.
16. Collect the crystals on a Buchner filter and wash with a small amount of cold
methanol. Allow the crystals to air dry and record the mass.
17. Compare the melting point of your isolated material with that of the crude cholesterol
starting material.
18. Obtain an IR of your isolated cholesterol.
Include a “Data Summary Table” as part of your Data/Results Section.
Here, the actual amount of reagents used should be specified. If the purity
of a reagent is not known, assume 100%. Solvents are generally not
necessary in this table. Note the use of ‘mmole’ in the table. It is generally
more convenient to use milli moles, since the numbers are generally easier
to manipulate. Your table should resemble the following.
Compound
MW
purity
cholesterol (crude)
bromine
zinc
dibromocholesterol
cholesterol (purified)
386.7
etc
etc
etc
etc
95%
etc
etc
etc
etc
wt used /
recovered
1.00g
etc
etc
etc
etc
Consider the following as part of your conclusion:
•
•
•
•
•
•
“100% wt”
0.95g
etc
etc
etc
etc
mmoles used
/ recovered
2.46
etc
etc
etc
etc
What is the underlying chemistry responsible for your observations in step 2 of
the procedure?
Why is cold methanol specified in step 5? What would be the effect if the
methanol was at room temperature
Occasionally a white solid is observed in steps 8-9 prior to the addition of water.
What might be the identity of the white solid?
What evidence (if any) do you have that the cholesterol isolated is more pure
than the initial cholesterol?
Are there any aspects of the procedure that are problematic? If you have
suggestions for improvements, mention them as part of the conclusion.
As part of your report, calculate the overall yield of purified cholesterol from
crude cholesterol. This should be reported in your “Data/Results” section and
reiterated in the Conclusion section.
Updated 6/20/2005