Epoxidation of Cholesterol Lab PDF

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Epoxidation of Cholesterol Lab...

Description

Epoxidation of Cholesterol

Date of Experiment: January 29, 2020 Date of Lab Report Submission: February 5, 2020

Objective: Epoxidation is a type of chemical reaction that transforms the c=c bond to an epoxide, by using a myriad of reagents such as air oxidation, hypochlorous acid, hydrogen peroxide, and

organic peracid. In this case, 3-Chloroperoxybenzoic acid reacted with cholesterol in order to form the epoxide form of cholesterol, 5α, 6α-epoxycholestan-3β-ol. The isolated product is then used for TLC.

Reaction Equations:

Theoretical Yield: N/A

Procedure:

Safety: Always wear safety goggles and nitrile gloves throughout the entirety of the lab. Lab coats should also be used throughout the lab with the correct shoes ( No open-toed shoes). All chemicals and waste must be disposed of in the right manner such as in the hazardous waste bin. No eating or drinking during the lab and make sure everything is cleaned up after the experiment. Place the pipettes containing alumina in the marked container. First, fill a 50mL beaker with around 30mL of water. Obtain a hot plate and turn the heat setting to 2 and if it is a digital hotplate then set it to 65°C. Obtain a scale and weigh out 0.100 g of cholesterol and place the cholesterol into a 10mL round bottom flask. Then, add 1mL of dichloromethane to the round bottom flask. Place the round bottom flask with dichloromethane in an empty 50mL beaker and make sure that it doesn’t fall off the benchtop. Obtain a test tube and place approximately 0.070g of 3-Chloroperoxybenzoic acid and dissolve it in the 1mL of dichloromethane. Get a hold of a Pasteur pipette and with caution add the dichloromethane/3-Chloroperoxybenzoic acid solution drop by drop into the round bottom flask. Then, add a boiling chip to the round bottom flask and connect the flask to a reflux condenser. The reflux condenser should have water running through it prior to applying heat. Before heating the contraption, make sure it has been checked by a lab technician. From the initial step, use the water bath as a heat source in order to reflux the mixture for around 15 minutes. Start the timer once the solvent begins to boil. After the 15 minutes are up, remove the reflux contraption from the bath of water. Then, pipette the mixture into a test tube and place it to the side. Get a hold of a new Pasteur pipette and push a small amount of glass wool into the pipette by using a boiling stick. Push the wool far enough into the pipette until there is a tight plug where the pipette narrows. Then, weigh out around 1.0g of alumina and add it to the pipette, if needed use a folded weight boat to help pour the powder. Go back to the area containing the alumina microcolumn pipette, the test tube rack filled with test tubes and a clean Pasteur pipette with bulb. Then, clamp the pipette so that a clean test tube in your rack is right below the pipette. Add the reaction mixture to the alumina column by using the clean Pasteur pipette and take your time to make sure the pipette doesn’t overflow. Slowly the solution from the reaction mixture will drain down into the pipette. Once the liquid is around the top of the aluminum in the column, start adding 7mL of Tert-Butyl Methyl Ether. Make sure the pipette doesn’t overflow but still add the ether

every time the level of the solution drops to around the top of the alumina. Keep adding the ether until the 7mL is gone and collect all the liquid that exits the column in a test tube. After all the liquid has been collected from the column, run a TLC plate by spotting the liquid from the column as well as a cholesterol standard. The solvent for the TLC jar must be Tert-Butyl Methyl Ether and not dichloromethane. Due to the fact that cholesterol and the product cannot be viewed on the plate under UV light, the stain phosphomolybdic acid (PMA) will be used. The stain can only be used under the supervision of a lab technician.

Observations: The experiment involved reacting 3-Chloroperoxybenzoic acid with cholesterol in order to form the epoxide form of cholesterol, 5α, 6α-epoxycholestan-3β-ol. This product was then isolated in order to use it for TLC. The epoxide and a cholesterol standard underwent TLC to determine which substance is polar. When pouring the alumina powder into the pipette, due to the small and fine particles some of the 1.0 g spilled and didn’t make it in. Also, some of the alumina stuck to the sides of the Pasteur pipette and therefore wasn’t used to plug the hole. Both issues could’ve been fixed and executed more precisely by using a small funnel or straw in order to get the alumina powder directly to the area where the pipette narrows.

Characterization: The Rf value is the amount that each mixture can travel and specifically is the ratio of the distance the spot of the compound moved above the origin to the distance the solvent moved above the origin. Since T-Butyl Methyl Ether is a non-polar solvent, the higher the mark goes, the more non-polar the substance must be.

Distance Traveled (cm)

Solvent Front (cm)

Rf Value

Epoxide Form of

3.8 & 2.5

5.7

Cholesterol Cholesterol Standard

0.667 & 0.439 (avg=0.553)

3.75

5.7

0.658

The Rf value for the epoxide was: 3.8/5.7= 0.667 & 2.5/5.7=0.439. The average of the two points were taken and it was determined to be 0.553. The Rf value for the cholesterol standard was: 3.75/5.7=0.658. Therefore, the cholesterol standard is more non-polar due to the fact that the mark is around 3.75cm from the origin. The experimental solution had two marks, one is 3.8 cm and another more prominent mark at 2.5cm away from the origin. Even though the first mark was slightly higher for the experiment, the mark was much lighter compared to the other mark at 2.5. This signifies that there must’ve been some impurities or deviations from the protocol that affected the results. Nonetheless, the mark demonstrates that the epoxide had an increase in polarity which is why the epoxide had a lower Rf value than the standard.

Conclusion: All in all, the experiment was executed with little problems that didn’t skew the results extremely. The objective of the experiment was to react 3-Chloroperoxybenzoic acid with

cholesterol to form the epoxide form of cholesterol, 5α, 6α-epoxycholestan-3β-ol. This isolated product should then undergo TLC with a cholesterol standard to determine whether or not the substance is polar or nonpolar. The TLC yielded trustworthy and valid results. The avg of the 2 marks from the epoxide TLC was 0.553 and for the cholesterol standard it was 0.658. So since the cholesterol standard had a higher mark it is more non-polar due to the fact that the solvent used was non-polar. In its pure form the epoxide form of cholesterol or 5α, 6α-epoxycholestan-3β-ol is polar so the results are reliable and accurate. The anomaly of the experiment is the small mark for the epoxide at 3.8cm. However, this mark was much lighter and smaller than the lower mark at 2.5cm which is where a polar substance such as the epoxide should be. If the experiment could be repeated again, a funnel or straw would be used to pour the alumina into the pasteur pipette so that the powder wouldn’t fall out or stick to the sides. This may be the reason why there is a mark for the epoxide at 3.8cm because the mark should be lower to signify the vast difference between nonpolar and polar substances.

Bibliography: “3-Chloroperbenzoic Acid 273031.” Meta-Chloroperbenzoic Acid, www.sigmaaldrich.com/catalog/product/aldrich/273031?lang=en®ion=US. “Dichloromethane.” Wikipedia, Wikimedia Foundation, 4 Feb. 2020, en.wikipedia.org/wiki/Dichloromethane. “Cholesterol.” Wikipedia, Wikimedia Foundation, 30 Jan. 2020, en.wikipedia.org/wiki/Cholesterol. “Alumina.” ChemSpider, www.chemspider.com/Chemical-Structure.14086.html. “Tert-Butyl Methyl Ether 306975.” (CH3)3COCH3, www.sigmaaldrich.com/catalog/product/sial/306975?lang=en®ion=US. “5,6α-Epoxy-5α-Cholestan-3β-Ol.” SpectraBase, spectrabase.com/compound/DGx94oGGRTV....