Amide reaction workup (Day 2)

Once the reaction is complete, the side products (a urea from DIC and protonated DIEA) need to be removed from the reaction along with any unreacted starting materials. After determining the reaction completeness with TLC, a series of aqueous washings will allow us to remove the unwanted compounds leaving only our protected biofilm inhibitor in the organic layer (see flowchart in Figure 1).

Figure 1. Flowchart demonstrating which wash will remove each impurity.
Figure 1. Flowchart demonstrating which wash will remove each impurity.

Monitoring the reaction

First, we will use Thin Layer Chromatography to determine if the reaction is complete.

  1. At the start of the lab period, conduct a co-spot Thin Layer Chromatography (TLC) experiment (read Padias text pg. 167-174, more instructions will be given in lab) comparing the reaction to the acid starting material.
  2. If the TLC experiment shows reaction completion, then move onto the workup. If the reaction is not yet complete, see Dr. Goode for help.

Washing away impurities

Now we will use the separatory funnel to remove the side products and any trace unreacted staring material.

  1. Remove the cap from your reaction and dilute it while it is still stirring with ethyl acetate (EtOAc) all the way to the top wide portion of the test tube (about 18 mL).
  2. After a minute, pour the entire solution into your separatory funnel. Be careful to only pour the liquid and not the stir bar. You may use another larger stirbar on the outside of the tube to help hold the smaller stirbar in place.
  3. Add 10 mL of 1 M citric acid solution to the test tube to rinse, and then pour this into the separatory funnel as well.
  4. Shake and vent as usual. The aqueous acidic solution will be on the bottom—drain it off into an Erlenmeyer flask and set aside.
  5. Add 10 mL of 1 M sodium bicarbonate solution (NaHCO3) to the separatory funnel. Shake and vent.
  6. Again, the aqueous layer (although basic this time) is on the bottom–drain it off into a separate flask and set aside.
  7. Last, add 10 mL of saturated sodium chloride (brine) solution to your separatory funnel. Shake and vent.
  8. Drain the aqueous layer out of the bottom into one of the other aqueous layers and set aside.
  9. Pour the organic layer out of the top of the separatory funnel into a clean Erlenmeyer flask. Add a scoop of sodium sulfate (Na2SO4) to the solution to help remove as much water as possible. Allow this to sit for 5 minutes.
  10. Decant (slowly pour so that you only get liquid and no solid) the solution into a clean, labeled, pre-weighed test tube. Place the test tube (open) into the collection rack

Cleaning Up

Once you have placed your dried organic layer in the collection rack, you can dispose of your aqueous layers down the drain while running the faucet. Clean all glassware with soap and water (use acetone only for stubborn grime). Clean up your hood, the area around any balances you used, and the common spaces near the reagents and waste containers.

After lab steps (done by Dr. Goode’s Research Lab)

After lab, the test tubes will be taken to the research lab and the solvent will be removed from each of your test tubes. This will leave a solid form of your product in the bottom of the test tube ready for the deprotection reaction.