Chem 113A Experiment E procedure (1)

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San Jose State University *

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113A

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Chemistry

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Dec 6, 2023

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Chem 113A Experiment E: Extraction and Purification of Alkaloids from Cinchona (Quinine and Derivatives) IMPORTANT!! Wear your PPE (safety eyewear, gloves and lab coats) for ALL parts of this experiment! Unless otherwise stated, all work must be performed in the hoods. Part I: Extractions and Filtrations IN THE HOOD – use the coffee grinder to break the Cinchona bark to small pieces (grind for 15-30 seconds) – the goal is to reduce it to granules (like sugar in the raw) not a fine powder! This may generate a lot of dust – avoid breathing this (wear gloves and keep your face mask on). Weigh about 10grams of the ground bark (again, be careful about the dust). Record the amount of ground bark in your notebook. Place the bark in a 50mL Erlenmeyer flask. Add approximately 20ml of 1% TFA until it covers the bark. Cover the flask with Duraseal, use a rubber band to hold it in place. Let the flask and contents sit at room temperature for 45 minutes. After 45 minutes, set up a vacuum filtration apparatus, check out a larger Buchner funnel from the stockroom. Transfer the filtrate to a 125mL Erlenmeyer flask. When the aqueous acid has all been filtered, return any solid to the original flask with the bark solids. Add sufficient aqueous acid, approximately 15-20 mL, to the bark solids to cover by ~1/4 inch of liquid. Record the amount of liquid you added. As before, use a rubber band to hold a cover of Duraseal TM in place. Let sit at room temperature for 45 minutes. Vacuum filter and combine with the first aqueous acid extraction into the same container. Record the volume of your crude aqueous extract. Cover the flask with Duraseal and a rubber band to hold it in place. Hand the flask to the instructor to store in the refrigerator until the next lab period. Part 2: Basification and Extraction of the neutral Alkaloids. Basification

For this part of the experiment, you will need 3M NaOH, pH paper, a graduate cylinder, separatory funnel and a glass rod or spatula. IN THE HOOD: Record the appearance of your solution before you begin. To your aqueous alkaloid extract, start by adding 2-10mL 3M NaOH. Do this carefully, 1mL at a time as you swirl your flask – you are mixing acid and base solutions. Check the pH by inserting a glass rod or spatula into the solution and touching the pH paper. Note any changes in the appearance of the solution. When you get closer to changing from acidic to basic, you may notice a change in the color of the pH paper. You will also notice a very obvious change in the appearance of the solution, especially when the pH paper indicates it has reached basic pH. Stop adding NaOH at this point and record your observations and the amount of aqueous base you used. Place your basified solution in your separatory funnel. Extract the aqueous solution 2x 25mL of ethyl acetate. Combine the ethyl acetate extracts and return to the separatory funnel. Wash the organic layer with 25mL of saturated sodium chloride. Dry the organic extract with magnesium sulfate. Weigh a clean and dry 125mL Erlenmeyer flask; record the weight. Gravity filter the dried ethyl acetate extract into the flask and hand to the instructor for the next period. Hopefully one of the alkaloids crystallizes during that time. In the next lab period observe the 125mL flask for crystal formation. Carefully decant the organic extract into a preweighed 100mL round bottom flask, rotavap the organic liquid. Obtain weight of the crude extract. Part 3: Column Chromatography and TLC Analysis Column Chromatography For this part of the experiment, you will need three short stem glass pipets, one long stem pipette, 10 small test tubes. IMPORTANT; avoid contact with silica gel, including breathing any of the fine dust. A face mask is strongly recommended for this step.

IN THE HOOD: To prepare the column first take a small piece cotton no bigger than a few rice grains and insert it into the glass pipet. Make sure it is securely at the neck of the pipet where the pipet starts to get smaller. Attach pipet to a ring stand using a clap. Then add silica gel to the pipet making sure to fill the pipet at least 2/3-3/4 full about 1 inch form the top of the pipet. Add the developing solvent, 20:70:4:6 dichloromethane:ethyl acetate:isopropanol:triethylamine to the pipet column. Make sure to have a beaker under the column to catch any solvent that may run through the column. Once the column is wet you make take it out of the hood and continue the work at your bench. Once the column is ready you will then prepare your sample by dissolving it in the minimum amount of solvent, no more than 0.5 mL. Place the tip of the pipette above the silica bed and gently squeeze, to allow the extract to flow down the side of the column to the top of the silica gel (don’t squeeze your pipette bulb too hard, or your extract will penetrate the top of the silica bed) Slowly add the sample to the column making sure not to disturb the silica gel. Let the solution with the alkaloids run into the silica gel. Once the liquid has been added to the silica bed add about 1 cm of sand to the top of the column. The sand forms a protective layer on top of the silica gel. When all the solution has been added to the column it is ready to start running. Start by adding solvent to the top of the column careful, again do not disturb the silica gel layer. Place a test tube below the pipet to collect the liquid running through the column into fractions. Label this test tube fraction 1, collect 3mL in fraction 1. When you have added 3ml of solvent, switch the test tube to fraction 2. Fractions 2-10 collect 1mL in each fraction. Continue to add solvent to the column making sure to never let the solvent level run below the silica gel line. Do not use more than 13 mL of solvent to run the column! Note any observations on the appearance of your column, such as colors. Label your test tube rack with your name and section # (your tubes should be numbered 1 - 10) and give it to your instructor. The developing solvent will evaporate by the next lab period.

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Analysis of the column fractions by TLC In the next lab period, make observations of the appearance in each tube. Add a few drops of the developing solvent to each tube. Add a few drops of the developing solvent to the ethyl acetate extract. Prepare a TLC plate and make 11 markings along the longest side. Label the first spot as “extract” and the rest from 1 to 10. Dissolve your samples in a few drops of methanol. Put your ethyl acetate extract on the first spot, and place the column fractions on spots 1 to 10. Develop the TLC place in a tank with the developing solvent. SAFETY: When the solvent reaches near the top of your TLC plate, remove it from the tank but DO NOT remove it from the hood until ~10min later; allow the solvent to completely evaporate before removing from the hood. View your TLC under the UV light and make a sketch in your notebook. Comment on any separation you observe and measure Rf values for spots that you see. If time permits, other methods of analyzing your column fractions may be performed.