Examining The Bromination And Debromination Of Cholesterol Biology Essay

Examining The Bromination And Debromination Of cholesterin Biology EssayBromination/debromination which is an important organic action that aims in purification of crude cholesterin from impurities which accept 3-cholestanol, 7-cholesten-3-ol, and 5,7-chlestadien-3-ol was performed in a laboratory scale for both weeks. Due to steric constraints, only cholesterin reacted with bromine and crystallized from the solution making it possible to be divide (Feiser, and Williamson 63). The dibromocholesterin formed is regenerated by reacting with Zinc dust. In addition, three methods were used to evaluate the posture. These were Sodium Iodide screen, argent Nitrate test, and Sulfuric acid test. NaI test showed a positive(p) response as color changed to yellow. The formation of the come in any case indicated a positive outgrowth. The sodium iodide reagent reacted with 1 and 2 alkyl halides finished an SN2 mechanism.On the divers(prenominal) hand, the silver nitrate reagent reacte d with 2o and 3 alkyl halides through an SN1 mechanism. Negative results were observed for both the commercial cholesterin and 1-chlorobutane (Zubrick 38). Conversely, the t-butyl chloride gave a positive result for the AgNO3test 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 tender loving care analysis affirm 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 come to the fore 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 steroid hormoneal compound found in both animals and plants. Despite the position 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.Figure 2 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 steroi d 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 thus reacted with zinc in order to regenerate pure cholesterol.Figure 1 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 running(a) 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 super C 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 flaskful. 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 bathe 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 scaremonger were turned on and gently heat until all the cholesterol dissolved in t-butylmethyl ether.Figure 3 Set-up for brominatio n reaction (Note the actual reaction mixture is not blue)The flask was removed from the water bath after all the cholesterol was whole 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.Figure 4 Set-up for the Addition of BromineThe water bath was replaced with codswallop and tap water and the reaction solution stirred intermittently with a glass stir rod for 10 minutes to complete the crystallization of the harvest-festival. or so 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 vanity filtration was done using a Buchner funnel shape 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 separatoryHYPERLINK http//community.acphs.edu/bbcswebdav/institution/Arts%20and%20Sci/Courses/CHE211/Common/lab/Organic-sde/BP-Dist/BP_General.html funnel in which 20 mL of deionized water was added, shaken and all owed 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 Fiv e test renders labeled A, B, C, D and E were used for this test. About 30 mg of the commercial cholesterol starting material was added to metro A 30 mg of dibromocholesterol to provide 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 severally 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 solut ion 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.Results Table 1 Percent Yield of synthesized cholesterolActual Yield0.29gTheoretical Yield1.08gPercent Yield26.9%Mass of Synthesized Dibromocholesterol was 1.57gTable 2 Melting Point TestCompoundTemperature CCommercial Cholesterol144.6-150.3Dibromochesterol105-109Synthesized Cholesterol147Table 3 Sodium Iodide testCompoundTime gyrusTempFirst Sign of ReactionCom. CholesterolNoneDibromocholesterol+ yellow(a)Synthesized Cholesterol+Chunky Yellow1-Chlorobutane+YellowTert-Butyl Chloride+CloudyTable 4 Silver Nitrate testCompoundTimeVortexTempFirst Sign of ReactionCom. CholesterolNoneDibromocholesterol+CloudySynthesized Cholesterol+Cloudy Yellow1-ChlorobutaneNoneTert-Butyl Chloride+CloudyTable 5 Sulfuric Acid TestCompoundTimeVortexTempFirst Sign of ReactionCom. Cholesterol+ColorDibromo cholesterol+ColorSynthesized Cholesterol+Color1-BromobutaneNoneTert-Butyl Chloride+ColorTable 6 Thin Layer Chromatography In 30% Ethyl Acetate/70% HexaneCommercial Cholesterol5.51.05.5Dibromocholesterol40.735.5Synthesized Cholesterol4.60.845.5DiscussionThe 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 deviance of the weight of the round bottom flask and the synthesized cholesterol by the synthesized cholesterols weight alone. The percent yield was calculated to be 26.9 percent. The synthesize process was not in effect(p) 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. T he 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 AgNO3test 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 value 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.