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THE PURIFICATION OF CHOLESTEROL BY BROMINATION AND DEBROMINATION Debra Jones

Abstract: The purpose of this experiment was to purify commercial cholesterol through bromination. It was calculated that 1.37g of purified cholesterol should have been extracted, however, only 0.269g was recovered. The percent yield was calculated to be 19.64%, while the percent error was found to be 80.36%. The actual amount of cholesterol obtained was much less than what it was calculated to be. Introduction: Halogenation is the reaction of an organic compound with a halogen molecule. The first step of this reaction creates a negative halide ion and a positive organic ion. After this step, the positively charged organic ion is added to the halide ion [1]. Halonium Ions are ions that have a halogen on it and carry a positive charge. They usually have an ionic nature, and an example of a halogen ion can be viewed in Figure 1[3].Vicinal dihalides are compounds that have halogens on adjacent carbons, while Geminal dihalides have both halogens on the same carbon. Trans vicinal dihalides have halide groups on the opposite faces of a carbon-carbon bond, while cis vicinal dihalides have halide groups on the same faces of the carbon-carbon bond. In general, the trans vicinal dihalides are more stable than the cis. The bonded groups are further apart and therefore have less repulsion. The stability of the dihalides F, Br, I and Cl is as follows: I is the strongest, followed by Br, then Cl, then F. The stability is due to the size and acidity of the atoms. Becaues I is the most stable, it is most likely to be synthesized, while F is the least stable, and is least likely to be synthesized[6].

Figure 1 An example of a halonium ion Bromination is used in the purification of cholesterol. Another method for cholesterol purification includes chromatography. First, the cholesterol was purified by using thin layer chromatography. Next, 5-10 mg of cholesterol was extracted by using methylene dichloride. Lastly, the cholesterol was measured by using gas chromatography. The column used was 3x1200 mm and the retention time was 38 minutes [7].

Halogenated compounds are environmentally important. Many xenobiotics are halogenated, such as antibiotics, pesticides, flame retardents, and other solvents. Halogenation and bromination are also important in the purification of compounds. The purpose of this experiment was to remove the 3β-cholestanol, 7-cholesten-3β-01, and the 5,7-cholestadien-3β-01 impurities from commercial cholesterol (Figure 2) [5].

Figure 2 Commercial cholesterol and its impurities

Methods 1.005 grams of commercial cholesterol was dissolved in 7 mL of ether by gently warming the solution. As the solution was warming, 5 mL of a bromine and sodium acetate in acetic acid solution was added. Once the solution began to crystalize, it was placed in an ice bath to cool. While still in the ice bath, the crystalizing solution was stirred for ten minutes in order to ensure complete crystallization occurred. While this solution was cooling, a new mixture of 3 mL of ether and 7 mL of acetic acid was cooled. After the crystals had cooled for ten minutes, the crystals were collected by using a Hirsch funnel. They were then washed with the cooled ether-acetic acid solution in order to remove excess mother liquor. The crystals were washed with methanol then transferred to a new flask. The solid was then weighed, and the mass was recorded. Results The datum collected from experimentation was the weight of the pure cholesterol solid. From the 1.005 g of impure cholesterol solid, 0.269 g of pure cholesterol solid was yielded (Figure 3). This result was calculated by subtracting the total mass of the pure solid, the filter paper, and the watch glass and subtracting the individual weights of the watch glass and the filter paper (Equation 1).

Equation 1: 

(Pure solid weight + Filter paper weight + Pure solid weight) – Watch glass weight – Filter paper weight = Pure Solid weight



33.101g -32.712g-0.120g = 0.269 g

Impure cholesterol weight: 1.005 g Watchglass weight: 32.712 g Filter paper weight: 0.120g Pure solid+ paper+ watch glass weight: 33.101g Pure solid weight: 0.269 g Figure 3 Mass of weighed substances used during experimentation. The impure cholesterol used in lab contained about 3% of impurities. To calculate theoretical yield, the weight of the cholesterol was divided by the molecular weight of cholesterol and then multiplied by the molecular weight of brominated cholesterol. (Equation 2). This yields the theoretical amount of pure cholesterol, which was determined to be 1.37g. Equation 2: 

Theoretical Yield= (Weight of cholesterol/MW cholesterol)x MW brominated cholesterol



Theoretical Yield = (0.975 g/ 386.65 g/mole) x 546.45 g



Theoretical Yeld = 1.37 g

From the theoretical yield of pure solid and the actual yield of pure solid, the percent yield can be calculated. This was done by dividing the actual yield by the theoretical yield (Equation3). The percent yield of pure cholesterol was calculated to be 19.64%, and the limiting reagent was determined to be cholesterol.

Equation 3:



Percent yield = (Actual Yield/Theoretical Yield) x 100



Percent Yield = (0.269 g/ 1.37 g) x 100



Percent Yield = 19.64%

Percent error was also calculated by using the actual and theoretical yields of the pure cholesterol. The actual yield was first subtracted from the theoretical yield. It was then divided by theoretical yield and multiplied by 100 (Equation 4). The percent error was calculated to be 80.36% Equation 4: 

Percent Error = [(Actual Yield – Theoretical Yield)/ Theoretical yield] X 100



Percent Error =[(0.269g-1.37)/1.37 g] x 100



Percent Error = 80.36%

Discussion: During experimentation, the impure cholesterol was dissolved in ether and heated in order to promote crystallization. Ether was chosen as the solvent because it was nonpolar, it had a relatively low boiling point (34.6⁰C), and it was stable [8]. These characteristics made it an ideal solvent for cholesterol because cholesterol is both nonpolar due to its long carbon chain and it has a very high boiling point (360⁰ C). Because both the solute and solvent are non-polar, therefore allowing cholesterol to easily dissolve in the ether. The high boiling point of cholesterol allows the solution to dissolve without any loss of product. Based on the experimentation of Atherton Seidell, it was determined that 100 cc. of ether dissolves 0.0007 gm of cholesterol at room temperature [9]. In the experiment, both acetic acid and sodium acetate were added to the cholesterol. The purpose of sodium acetate and acetic acid was to act as a buffer solution.

A buffer is and aqueous solution that is composed of a weak acid or base its corresponding conjugate base or acid. In this experiment, acetic acid was the weak acid, and sodium acetate was its corresponding conjugate base. The purpose of this solution was to buffer bromination [10]. The mechanism of the bromination of cholesterol is a multi-step process that involves theᴨ electron cloud of the alkene acting as the nucleophile and bromine acting as the electrophile [4]. A nucleophile is a negatively charged compound that “attacks” an electrophile to donate an electron pair in order to form a chemical bond [6]. An electrophile is a positively charged reagent that is attracted to and accepts an electron pair in order to bond to a nucleophile [6]. After the nucleophile attacked the electrophile, the sigma bonds in the Br2 are broken, causing the negative bromide ion to leave. This created an intermediate where the two molecules form a tertiary carbocation through the sharing of their non-bonding electrons. Another reaction with an additional halide completes the reaction process. The intermediate carbocation formed dibromocholesterol after being attacked from behind by a bromide ion. The ion is formed when the ᴨ electrons combine in the empty Br+ P orbital. This new molecule has bromine molecules located in both the trans and diaxial configuration (Figure 4) [4]. In bromination, there can be anti-addition and syn-addition. Anti-addition is a reaction in which the bromine ions add on to opposite faces of a carbon-carbon bond. Syn-addition is when during the reaction the bromine ions add onto the same face of a carbon-carbon bond. In this reaction, the addition of bromine ions was syn-addition. The bromine molecules are on opposite faces of the carbon-carbon bond. The resulting configuration of the cholesterol molecule was tran vicinal dihalide, meaning that the bromine molecules are on opposite sides of adjacent carbons. In general, the trans configuration is more stable than the cis formation (bromine ions on the same side) due the groups bonded to the ᴨ bond being further apart, and thus leading to less repulsion [6]. Despite the lack of a double bond, trans formation is still possible around the single bond. This is due to the sigma bond being located along the axis of the bond. This allows

the surrounding orbitals to be symmetrical around the axis, which leads to the bond rotating, due to there being no energy barrier [6].

Figure 4 The mechanism of the bromination of cholesterol. Racemization is the converting of an optically active substance that is enantiomerically pure into an optically inactive mixture that has more than one enantiomer present [11]. The product of this experiment is not racemic because it is enantiomerically pure. The products are not super-imposable on each other. During experimentation, it was found that the bromonium ion is much more stable that the carbocation. This can be attributed to the resonance within the bromonium ion. In a carbocation, the stabilizing factor of the molecule is its inductive bonds. In bromonium ions the stabilizing agent is its resonance, which causes it to form a much stronger, more stable bond [2]. During the experiment, the solution was cooled in an ice bath. This part of the procedure was done to ensure that there was full crystallization of cholesterol dibromide. Another part of the procedure was ether and acetic acid wash. The purpose of the ether and acetic acid wash was to remove the excess yellow mother liquid. This helps ensure the purity of the crystals. For the collection of crystals in macroscale bromination, a Hirsch funnel was used to extract the crystals, however there are other methods for crystal extraction. A Wilfilter is a device that is used to collect small quantities of crystals. It is placed upside-down in a centrifuge tube spun in a centrifuge in order to

collect the crystals [12]. It would me most useful in microscale bromination because there would be less product. When washing the crystals ethanol was used because bromide is a stronger nucleophile towards the intermediate than ethanol, therefore ethanol does not carry out any reactions with the intermediate. However, if it did react with the intermediate, it would form peroxides (Figure 5) [8].

Figure 5 The reaction between ether and the intermediate to form peroxides. Debromonation is a reaction that removes bromine from a compound [6]. Although debromonation was not performed in this experiment, the purpose of the reaction would be to obtain completely pure cholesterol. In order for debromination to occur, zinc dust would be added to the crystals. From the experiment, the percent yield was determined to be 19.64% and percent error was 80.36%. Some possible errors that could have occurred during experimentation could include not allowing the crystalizing solution to sit in the ice bath for long enough. This would not have allowed enough time for full crystallization, causing less product to be collected. Another possible source of error could be that when the crystals and filter paper were being removed from the funnel, some of the crystals could have become dislodged from the filter paper and left in the funnel. This would also cause less product to be collected. To avoid such errors in the future, extra care must be taken when carrying out the procedure.

Conclusion: This experiment yielded 0.269 g of cholesterol. The theoretical yield was calculated to be 1.37 g. From the actual and theoretical yield the percent yield and the percent error were calculated to be 19.64% and 80.63% respectively. The actual yield of the cholesterol was much less than that of the theoretical yield. Further experimentation can be done to attempt to narrow the gap between the theoretical and actual values. The bromination and halogenation techniques can also be used for the purification of other various compounds in future experiments.

References: [1] Roberts I, Kimball GE. The Halogenation of Ethylenes. J Am Chem Soc 59(5): 947-948, 1937. [2] Vyas PV, Bhatt AK, Ramachandraiah G, Bedekar AV. Environmentally benign chlorination and bromination of aromatic amines, hydrocarbons, and naphthols. ELSEVIER 44(21): 4085-4088, 2003. [3] Olah GA, Bollinger M. Stable Carbonium Ions. XLVIII. Halonium Ion Formation via Neighboring Halogen Participation. Journal of the American Chemical Society 89 (18),1967. [4] McMurry, John. Organic Chemistry, Enhanced Edition. Vol. 1. Pacific Grove, CA: Brooks/Cole Pub., 1992. Print. [5] Chaudhry GR, Chapalamadugu S. Biodegradation of halogenated organic compounds. Microbiology and Molecular Biology Reviews 55(1): 59-79, 1991. [6] Klein, David R. Organic Chemistry I as a Second Language. New Jersey: John Wiley, 2008. Print.

[7] Shikita M, Hall PF. Cytochrome P-450 from Bovine Adrenocortical Mitochondria: an Enzye for the Side Chain Cleavage of Cholesterol. The Journal of Biological Chemistry 248: 5598-5604, 1973. [8] Thistle JL, May GR, Bender CE, Willams HJ, LeRoy AJ, Nelson PE, Peine CJ, Petersen BT, McCullough JE. Dissolution of Cholesterol Gallbladder Stones by Methyl Tert-Butyl Ether Administered by Percutaneoud Transhepatic Catheter. The New England Journal of Medicine 320: 633-639, 1989. [9] Seidell, Atherton. Solubilities of Inorganic and Organic Compounds; a Compilation of Quantitative Solubility Data from the Periodical Literature. New York: Van Nostrand, 1919. Print. [10] Fieser LF. Cholesterol, ∆5-Cholesten-3-one, and ∆4-Cholesten-3-one. Organic Syntheses 35(43), 1955. [11] Kim MJ, Kim WH, Han K, Choi YK, Park J. Dynamic Kinetic Resolution of Primary Amines with a Recyclable Pd Nanocatalyst for Racemization. Organic Letters 9(6):11571159, 2007. [12] Williamson, Kenneth L. Techniques Labs for Macroscale and Microscale Organic Experiments. Belmont, CA: Brooks/Cole, 2012. Print.

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