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Debromination and Bromination of Cholesterol

Bromination/debromination which is an important organic reaction that aims in purification of crude cholesterol from impurities which include 3-cholestanol, 7-cholesten-3-ol, and 5,7-chlestadien-3-ol was performed in a laboratory scale for two weeks. Due to steric constraints, only cholesterol reacted with bromine and crystallized from the solution making it possible to be separated (Feiser, and Williamson 63). The dibromocholesterol formed is regenerated by reacting with Zinc dust. In addition, three methods were used to evaluate the effectiveness. These were Sodium Iodide test, Silver Nitrate test, and Sulfuric acid test. NaI test showed a positive response as color changed to yellow. The formation of the precipitate also indicated a positive result. The sodium iodide reagent reacted with 1° and 2° alkyl halides through an SN2 mechanism. On the other hand, the silver nitrate reagent reacted with 2° and 3° alkyl halides through an SN1 mechanism. Negative results were observed for both the commercial cholesterol and 1-chlorobutane (Zubrick 38). Conversely, the t-butyl chloride gave a positive result for the AgNO3 test and a negative result for the NaI test. The synthesized cholesterol was 0.29 gram and the theoretical yield was 1.08 gram. This gave a percent yield of 26.9 gram. Although this was a low yield the TLC analysis confirmed a high purity of the synthesized cholesterol. The melting point of the synthesized cholesterol and commercial cholesterol seems to fall in the same range. This confirms the purity of the synthesized cholesterol. TLC analysis was carried out to confirm the purity of the analysis. The distance traveled by commercial cholesterol was 5.5 whereas that travelled by the synthesized cholesterol was 4.6. The absence of other spots on the TLC plate confirms that there were no contaminations present in the sample.
Introduction
Cholesterol is an important steroidal compound found in both animals and plants. Despite the fact that cholesterol causes diseases, it plays a vital role in life. For example, cholesterol is the main structural component in cell walls and in myelin sheath formation. It is also a major precursor for most steroid hormones. Crude cholesterol is isolated from natural sources and various methods have been used in its purification. Crude cholesterol contains approximately 3-5% contamination. Some of the contaminants are 3-cholestanol, 7-cholesten-3-ol, and 5,7-chlestadien-3-ol shown below.
Some common contaminants of commercial cholesterol The main objective of this experiment was to purify commercial cholesterol using organic reaction chemistry, including the use of the electrophilic addition. For complete purification of cholesterol from the above impurities is achieved by a reaction of bromine with cholesterol to generate dibromocholesterol. Because of the steroid ring structure present in these compounds that causes steric constraints, only cholesterol reacts with bromine to form an insoluble diaxial dibromo compound through electrophilic addition. On the other hand, cholestanal does not react with bromine and the other two contaminants are dehydrogenated by bromine leading to formation of soluble dienes and trienes respectively. The dibromo-cholesterol precipitates as a solid leaving the other impurities in the reaction solvent. A purification step such as solvent washing or crystallization is carried out to separate the solid from the impurities. The solid dibromo-cholesterol is then reacted with zinc in order to regenerate pure cholesterol.
Reaction Scheme for the Bromination/Debromination of Cholesterol
To test the effectiveness of this reaction three different chemical tests namely sodium iodide in acetone, silver nitrate in ethanol test, and sulfuric acid test, were performed. Each of these tests is selective for a specific functional group. Dibromocholesterol contains both primary and secondary alkyl halides and reacts with a sodium iodide in acetone and silver nitrate in ethanol to form a precipitate or a cloudy solution. In addition, the presence of double bonds in dibromocholesterol in form of alkene makes it possible for the formation of a fluorescent green sulfuric acid layer and a red chloroform layer when reacted with sulfuric acid (Landgrebe 78).
Material and Methods
1g of commercial cholesterol was added to a 25 mL Erlenmeyer flask. 7 mL t-butylmethyl ether was measured with a graduated cylinder and added to the flask containing the cholesterol and a magnetic stir bar. A water bath was then set up on the hotplate in the hood. The Erlenmeyer flask contain the reaction solution was inserted into the water bath and clamped as shown below. The heat and the stirrer were turned on and gently heat until all the cholesterol dissolved in t-butylmethyl ether.
The flask was removed from the water bath after all the cholesterol was completely dissolved and allowed to cool to room temperature. After the cholesterol solution was cooled, the flask was clamped to the ring on the hot plate as shown in figure 4 and stirred without heating. A burette was then used to dispense 5 mL of bromine solution into the flask. A precipitate solution formed almost immediately.
The water bath was replaced with ice and tap water and the reaction solution stirred intermittently with a glass stir rod for ~ 10 minutes to complete the crystallization of the product. About 20 mL of the t-butylmethyl ether – acetic acid solution was then dispensed in a clean 50 mL Erlenmeyer flask which was clamped to a ring stand and allowed to cool in the ice bath. A vacuum filtration was done using a Buchner funnel and filter paper. The solid in the filter was washed using ~10 mL of the cooled solution of t-butylmethyl ether – acetic acid and then with ~10 ml of methanol. The solid was then allowed to dry with the vacuum on for about 5 minutes. the dibromocholesterol melting point was measured and recorded. The dry solid was weighted and sealed in a vial and stored for next experiment. To debrominate cholesterol, 20 mL of t-butylmethyl ether, 5 mL of acetic acid and 0.2 g of Zn dust were added into the Erlenmeyer flask containing the dibromocholesterol solid. The mixture was swirled for 5-10 minutes in the hood and sonicated in 5 minutes to allow the reaction to go completion. After sonication the solids present were removed by gravity filtration method into a clean 125 mL Erlenmeyer flask. The filtrate was transferred to a 125 mL separatory funnel in which 20 mL of deionized water was added, shaken and allowed to separate into layers. The two layers formed were then separated as water layers and organic (ether) layers. The ether layer was washed with 20 mL of 10% NaOH and then 20 mL of saturated NaCl solution. 100mg of the drying agent magnesium sulfate was added to the organic layer and the solution swirled until dry. The drying agent was removed by gravity filtration using a glass funnel fluted filter paper and a very dry 50 mL Erlenmeyer flask. The flask was placed in a warm water bath and then ice cooled for 10 minutes until all but 5 mL of the ether remained following a precipitate formation from the solution. The remaining solvent was decanted and the synthesized cholesterol transferred and allowed to dry in the hood for 20 minutes.The dry solid was weighed and the weight recorded. In addition the melting point was also taken and recorded. To evaluate the effectiveness of the bromination reaction three chemical reactions mentioned above were carried out. NaI in acetone test Five test tubes labeled A, B, C, D and E were used for this test. About 30 mg of the commercial cholesterol starting material was added to tube A; ~30 mg of dibromocholesterol to tube B; ~30 mg of the synthesized cholesterol product to tube C; ~0.3 mL of 1-chlorobutane to tube D; and ~ 0.3 mL of t-butyl chloride to tube E. In addition, about 3 mL of acetone was added to each tube to completely dissolve all the compounds. Solutions A-E was used to do the NaI in acetone test as well as the AgNO3 in ethanol test. Tubes A-C did the TLC as well. The NaI in Acetone and AgNO3 in Ethanol tests were performed by setting up a test tube rack containing ten small test tubes. The test tubes were labeled N1 – N5 and A1 – A5. 1 mL of NaI in acetone reagent was added to test tubes N1 – N5, and 1 mL of AgNO3 in ethanol reagent to test tubes A1 – A5. This was followed by adding 5-8 drops of A solution to test tube N1 and tube A1, 5-8 drops of solution B to test tube N2 and tube A2, 5-8 drops of solution C to to test tube N3 and tube A3, 5-8 drops of solution C to test tube N4 and tube A4, and 5-8 drops of solution C to test tube N5 and tube A5. The test tubes were heated for a while and all the observations recorded. The sulfuric acid for alkenes test was performed by additional solutions of cholesterol and dibromocholesterol with five dry-cleaned test tubes 1-5. 10 mg of commercial cholesterol was placed in tube 1, ~10mg of the dibromocholesterol to tube 2 ~10 mg of your synthesized cholesterol to tube 3, ~10 mg of 2-chlorobutane to tube 4, and 10 mg of cyclohexene to tube 5. About 1 mL of chloroform (CHCl3) was added to each tube and vortex to completely dissolve all solids. In addition, 0.5 mL of H2SO4 was then added to each tube. The observation for this reaction was recorded in the notebook. The TLC analysis of cholesterol and dibromocholesterol was performed by obtaining a silica gel TLC plate and setting it up to run TLC analysis on solutions A-C above. The plate was spotted with each solution and developed by placing the plate using 30% ethyl acetate: 70% hexane as the mobile phase. The developed plates were viewed under UV lamp and in the I2 chamber and observations recorded.
Discussion
The yield of the synthesized cholesterol was .29 grams (Table 1). The theoretical yield was 1.08 grams. The actual yield was calculated by taking the difference of the weight of the round bottom flask and the synthesized cholesterol by the synthesized cholesterol’s weight alone. The percent yield was calculated to be 26.9 percent. The synthesize process was not efficient due to the low yield and percent yield of the synthesized cholesterol.
The melting point of the synthesized cholesterol and commercial cholesterol seems to fall in the same range. This confirms the purity of the synthesized cholesterol. NaI test showed a positive response as color changed to yellow. The formation of the precipitate also indicated a positive result. The sodium iodide reagent reacted with 1° and 2° alkyl halides through an SN2 mechanism. On the other hand, the silver nitrate reagent reacted with 2o and 3° alkyl halides through an SN1 mechanism. Negative results were observed for both the commercial cholesterol and 1-chlorobutane. Conversely, the t-butyl chloride gave a positive result for the AgNO3 test and a negative result for the NaI test. The stationary phase of the TLC test was the silica gel TLC plate and the mobile phase was 30% Ethyl Acetate/70% Hexane (Table 6). The distance traveled by commercial cholesterol was 5.5, and for the synthesized cholesterol was 4.6. The difference in the distance traveled and the Rf values of the samples commercial and synthesized cholesterol were pure. Since there were no other spots visible on the TLC plate was a clear indication that there were no contaminations of other chemical compounds present in the sample.

Changing Concentration of Hydrochloric Acid

How will changing the concentration of hydrochloric (HCl) acid affect the rate of hydrogen gas (H2) production during the reaction with magnesium (Mg), using the pressure buildup by hydrogen gas?
Introduction Factors that influence rates of reactions include change in concentration, temperature, surface area, or the addition of a catalyst. This experiment will specifically investigate the effect of concentration change of the reactants upon the rate of reaction, using hydrochloric acid and magnesium strip. The concentration of HCl acid solution is controlled through serial dilution.
2HCl(aq) Mg(s) → MgCl2(aq) H2(g)
This experiment in particular will explore how the pressure changes as the above reaction proceeds. Because the reaction produces hydrogen gas as a product, building up more pressure within the confined space of a test tube, a pressure sensor will measure the rate of reaction. After the reaction begins, approximately 20 seconds of data will be collected with each trial, in order to formulate a common trend (a graph of pressure over time). With average slopes of different amount of concentrations, a linear regression line will then be created to sketch the trend, regarding the effect of concentration upon pressure-the rate of reaction.
Hypothesis According to Collision Theory, the reactant particles must collide together, and thus creating a reaction. Because increasing the concentration of HCl acid solution also means an increase in the number of hydrogen and chloride ions, collision between the reactant particles increases as well, resulting in more products-hydrogen gas. With more production of hydrogen gas in the confined test tube, pressure will build up.
Therefore, if-at a given period of time-the concentration of HCl acid solution increases, then the rate of reaction will increase accordingly, because more collisions will occur, producing hydrogen gas at a higher rate.
Variables
Variable description
Method of measuring variable
Dependent variable
Pressure buildup due to the reaction between hydrochloric acid solution and magnesium
During the reaction, H2gas is produced, thus increasing the volume within the confined space of a test tube and increasing the pressure. This change will be recorded by a pressure sensor. Collecting data for about 10 seconds before the injection of the magnesium strip, the measurement of pressure will continue for about 20 seconds after the reaction begins. Three trials are required for each concentration of HCl solution to minimize random error.
Rate of reaction
Using the more accurate initial rate of the reaction, about 10 seconds of the graph after the reaction begins will be used to create a slope of change in pressure over time.
Independent variable Concentration of HCl solution
Using serial dilution along with apparatus such as micropipette and flask, the 1M hydrochloric acid solution will be diluted into 0.5M, 0.25M, 0.125M, and 0.0625M.
Controlled variables Mg strip (length)
Using a ruler and scissor, the Mg strip will be cut into 15 pieces, each being 1cm.
Volume of hydrochloric acid solution
For each concentration, 3cm3of hydrochloric acid solution is used, accurately measured by a pipette.
Temperature of reactants
The temperature remains constant at room temperature (approx. 25 degrees Celsius) throughout the entire experiment.
Shaking of the test tube
To create the most accurate results possible, physical motion when slightly shaking the test tubes must be repetitive in the same way for each trial.
Size of the test tube
Because different sizes of test tubes would mean different volumes as well, constant size (volume) is essential, preferably small so that the reaction will be more conspicuous. To do this, 15 identical test tubes are used.
Table 1: List of Variables
Apparatus and Materials 1M hydrochloric acid solution
Distilled water
Magnesium strip
Flask (50cm3)
Pressure sensor
Logger Pro
Micropipette
15 identical test tubes
Sandpaper
Scissor
Procedure Put 20cm3 of 1M HCL solution in the flask and dilute it to 0.5M with 20cm3 of distilled water.
Using the serial dilution as in step 1, prepare 10cm3 solutions with concentrations of 1M, 0.5M, 0.25M, 0.125M, and 0.0625M.
Add 3 cm3 of each solution into labeled test tubes using the micropipette.
Repeat step 3 to prepare three test tubes of each solution (15 in total)
Cut out the magnesium strip into 15 pieces of 1cm and sand them with sandpaper.
Put the cut out magnesium strip into the test tube with 1M HCl solution.
Then quickly cover the test tube with the pressure sensor.
Start collecting data while shaking the test tube in a consistent manner for about 25 seconds after the reaction begins.
Repeat steps 6 to 8 for all other test tubes.
Data Collection and Processing

Qualitative Data
After the injection of the magnesium strip into the HCl solution, it effervesces and pressure inside the test tube begins to build up.
With test tubes of higher concentration, the pressure seems to be higher within the time limit and more bubbles form.
At the end of the reaction, the solution’s color changed to transparent yellow.
The reacted solution (product) gives off a foul smell.
Processed Data

Data Presentation 1 0.5 0.25 0.125 0.0625
Uncertainties Standard Deviation
Standard deviation was calculated and represented in the rate of reaction vs. concentration graph as error bars.

Standard deviation for different concentration of HCl solution
Standard deviation was calculated by a graphing calculator.
Uncertainty due to the serial dilution of HCl solution
Uncertainty due to 1cm3
Uncertainty during dilution

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