Final Workup

Now that the carboxylic acid functional group has been restored, we will use its acid/base reactivity to purify our target product. We will first extract the product out of the organic layer and into the aqueous layer by deprotonating the carboxylic acid to the negatively charged carboxylate (see Scheme 1). Then, we will acidify the aqueous layer to add the proton back onto the carboxylate and restore the carboxylic acid, which is now more soluble in organic solvent that water. A final extraction of the carboxylic acid back into an organic layer provides our purified product.

Procedures

You will need to begin by collecting your labeled reaction tube. Make notes in your notebook about how the product solid looks prior to starting the extraction.

1. Dissolve your solid in 15 mL of Ethyl Acetate and transfer this organic layer to your separatory funnel. Rinse your test tube with another 5 mL of Ethyl Acetate and add it to the separatory funnel. Your initial organic layer will be 20 mL.
2. Extract the product out of the organic layer. Start with 10 mL of 1 M NaHCO₃. The organic layer will be the top layer, and the basic aqueous layer will be on the bottom. Shake and vent as usual. Then collect the aqueous (bottom) layer into a clean 125 mL Erlenmeyer flask. Be sure the flask is labeled as “Aqueous Extract.”
3. Repeat step 2 two more times and combine the aqueous layers together (for a total of 3 extractions). At the end of the last extraction, you will have 30 mL of the basic aqueous solution in the Erlenmeyer flask. This solution now contains your deprotonated product.
4. Now pour the organic layer into a different 125 mL Erlenmeyer flask that is labeled “Organic Layer 1.” Set this to the side for now. This organic solution should now only contain any impurities left over from the deprotection reaction.
5. Carefully and slowly add 10 mL of 3M HCl to your aqueous solution. As you add HCl, the excess bicarbonate will be converted to salt, water, and CO₂ gas and form lots of bubbles. Your product should also be protonated in this process and form a solid suspension in the aqueous layer (making it cloudy). Check the pH using pH paper–you are looking for a pH that is less than 4. If the pH is too high, add more 3M HCl 1mL at a time until you get there. If your pH is less than 4, then proceed to the next step. Note: if your amino acid is Lysine, see Dr. Goode before adding acid.
6. Pour the acidified aqueous layer into the separatory funnel. Then add 10 mL of Ethyl Acetate, which should stay on the top. Shake and vent as usual. The solid should dissolve in the Ethyl Acetate layer as you mix. After mixing, dispense the bottom layer through the stopcock into the “Aqueous Extract” Erlenmeyer flask. Then pour the top layer into a clean Erlenmeyer flask labeled “Organic Layer 2: Product.” Your separatory funnel should be empty.
7. Repeat step 6 completely one more time. At the end of the repeat, you should have an aqueous layer in one Erlenmeyer Flask labeled “Aqueous Extract” and 20 mL of an Ethyl Acetate solution in the Erlenmeyer Flask labeled “Organic Layer 2: Product.”
8. Dry the organic layer by adding 2 heaping scoops of Sodium Sulfate (Na₂SO₄) to your Organic Layer 2. Allow this to sit for 5 minutes.
9. Now decant your solution into a pre-weighed large vial (provided by Dr. Goode). Cap the vial and label it with the compound code (Three letter code of amino acid-number of carboxylic acid, i.e. Phe-69), the mass of the empty vial, and your name. Turn this into the designated area.

Cleaning up

Once you have turned in your vial containing your product (Organic Layer 2), you can clean up the others. The Aqueous Extract now only contains an acidic salt solution, which can be discareded down the drain with running water. The “Organic Layer 1” should be poured into the waste container in the corner hood of the lab. The sodium sulfate crystals can be dissolved in water and also washed down the drain with water. Clean all glassware with water first, and then with acetone (into the waste container) if needed.