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Bromination of Cholesterol and Debromination: Purification of Cholesterol

Bromination of Cholesterol and Debromination: Purification of Cholesterol

Written by: Nicholas Connor

The purpose of this experiment was to understand alkene-halide reactions by purifying commercial cholesterol.

Analysis

The purpose of this experiment was to remove the impurities present in commercial cholesterol; to yield pure 5-Cholesten-3B-ol. Bromination was the method chosen to remove traces of 3B-cholestanol, 7-cholesten-3b-ol, and 5,7-cholestadien-3B-ol that were assumed to be present in the sample used. The net reaction of bromination follows:

To begin removing the impurities, the cholesterol sample (0.97 g) was dissolved in tert-butyl-methyl-ether (7mL).  This ether does not dissolve cholesterol dibromide as well as the impurities, making it a decent purification solvent. Addition of acetic acid and bromine (5mL) initiated the bromination; the alkene of cholesterol nucleophilically attacked bromine and formed tertiary a carbocation. Subsequently, the bromide ion made a backside attack to create the desired product.

The reaction seemed to take place almost immediately and the cholesterol dibromide rapidly crystallized. To complete crystallization, the now yellowish ether solution was placed in an ice bath for twenty minutes and followed by vacuum filtration and washing. A sparse-yellow tint was observable in the product and some was lost in the mother liquor.

Bromination of Cholesterol Mechanism

After filtration from the mother liquor, the cholesterol dibromide was theoretically stripped of all its original impurities. In order to return to a non-halogenated product, a debromination reaction was conducted using zinc.

The crystallized cholesterol dibromide was dissolved (after ~2 minutes) in tert-butyl-methyl-ether (20 mL) and acetic acid (5 mL). Zinc dust (0.2 g) was added to the solution as well. Several minutes later, the reaction was apparent and zinc acetate began precipitating, resulting in a cloudy solution. Not enough ether was used and some cholesterol also precipitated. The solution was decanted into a separatory funnel, removing the zinc dust; some cholesterol was lost. Addition of water dissolved the zinc acetate and sodium hydroxide (3M) removed the remaining acetic acid. Saturated sodium chloride solution was also added to reduce water content. The ethereal solution was separated from the aqueous layer. During the separation, cholesterol penetrated the funnel’s working parts and some product was lost. The ether solution was dried and then evaporated to crystallize the final product (0.76 g, see Table A below).

Theoretical yield
0.97 g

Experimental yield
0.76 g

Percent Yield
78%

Literal Melting Point (C)
149-150

Experimental Melting Point (C)
120.1-135.8

Percent Error
16%

Table A: Experimental Results

In conclusion, the experiment was fairly effective at purifying commercial cholesterol. The product was not as pure as expected, and shows 16% discrepancy with accepted melting point values. Melting point was assessed seven days after the experiment, and by that time the white product accumulated a significant brown color. Perhaps the product was contaminated after the crystallization, as it was resting in open air. Contamination would explain the low melting point results. Furthermore, the procedure may be improved by using 25 mL of the ether during the debromination and by adding 5-10mL of water after the zinc acetate precipitation. Otherwise, the yield was quite impressive (see Table A), considering the divisions lost in the mother liquor (during bromination) and separatory funnel (in debromination). Successive attempts would surely improve yield and purity.

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