Project 4 Procedure

Week 1: Part A

To a mixture of glycine (10 mmol) and potassium hydroxide (10 mmol) in methanol (7 mL), add 2,4-pentanedione (10 mmol). Reflux the mixture for 1 hour. (Lightly grease the joint between the flask and the condenser.) After the reflux period, cool the flask in a ice-water bath, then pour the mixture into ice-cold acetone (30 mL). Vacuum filter the resulting solid, and allow it to dry under vacuum for at least 30 minutes. Record the mass of the product. Obtain an IR spectrum and ¹H and ¹³C NMR spectra (solvent: D2O). Save your product in a labeled vial for Part B.

Reminders & Hints

1. You must complete the Project 4A Prelab Quiz before entering the lab to perform this procedure.

2. Strongly basic conditions can etch glassware and cause joints to stick together. The grease will prevent this from happening in your apparatus. Use a VERY light coating of grease on the condenser joint. At the end of your reaction, wipe down the condenser joint with acetone to remove the grease.

Week 2: Part A Analysis Workshop

Come to lab prepared to discuss the NMR analysis of your product from Week 1. Lab will be a discussion-based workshop to help you better understand the  product chemistry and NMR data.

Week 3: Part B

Reflux a mixture of the Part A product (5 mmol), triethylamine (35 mmol), and acetic anhydride (159 mmol) for 30 minutes. Cool the mixture to room temperature, then add ~50 mL ethyl acetate to dissolve any solids. Wash the solution with 10% sodium hydroxide (2 × 40 mL) followed by brine (20 mL). Dry the organic layer over sodium sulfate. Gravity filter the mixture and remove the solvent by rotary evaporation. Isolate the product by column chromatography using a large diameter column, ~30 mL silica gel, and a 9:1 petroleum ether:acetone mobile phase. Recombine only fractions containing the product and remove the solvent by rotary evaporation. Obtain GC-MS, IR, and NMR (CDCl3) data for the product.

Dr. O’Neal is collecting data on the success of Project 4. After you have isolated your products, please report your yields and any comments in this data table.

Reminders & Hints

1. You must complete the Project 4B Prelab Quiz before entering the lab to perform this procedure.

2. Use your time wisely! Go ahead and set up your TLC materials and chromatography column during the reflux period!

3. If your solids do not dissolve upon addition of ethyl acetate, add a little of the NaOH solution to your flask. This should dissolve any remaining solids.

4. You should TLC the crude product you obtain after extraction to get a sense of what to expect during column chromatography. Timesaver: TLC the ethyl acetate solution  and allow the plate to run while the mixture is on the rotary evaporator. (You don’t need to spot anything else on the TLC plate). The product is visualized by UV.

5.  Timesaver: The procedure indicates that that the crude ethyl acetate solution is washed with an aqueous solution three separate times. The organic layer can stay in the separatory funnel for this entire process! Just wash it with the appropriate solution and then allow the aqueous layer to drain from the funnel.

3. To refresh your memory on column chromatography, review the technique video here. In addition, a step-by-step example procedure is given below. For this experiment, your silica should be about 1/3-1/2 the height of the plastic column.

Column Chromatography Step-by-Step

1. Build the chromatography column:

a. In a 150 mL beaker, mix about 30 mL of silica gel with 9:1 petroleum ether:acetone until you have a slurry (melted milkshake consistency).

b. Clamp the column so that it is vertical, place an Erlenmeyer flask beneath it (to collect solvent that passes through), and add the slurry of silica. Allow the solids to settle for a few minutes.

c. Using a Pasteur pipet, add extra solvent to wash down the walls of the column. Use a swirling motion to add the solvent to the walls of the column in order to prevent disturbing the column integrity. Tap the column to make the top of the silica even.

d. Allow the solvent to drain until it reaches the top of the column.

e. Add a small layer (~0.5 cm) of sand on top of the silica. Wash down any sand from the walls of the column with solvent using the same swirling motion.

f. Use a Pasteur pipet to add a few milliliters of solvent to the top of the column. Drain until it reaches the top of the column.

2. Using a Pasteur pipet, add your reaction sample to the column using the same swirling/gentle addition method described above. Remember that you want a tight band of your sample at the top of the column. Add your sample in a single shot and do NOT try to wash in residue with additional solvent. Allow the sample to seep into the column.

3. Add the mobile phase to the top of the column: Start with just a thin layer and allow that pass into the column. (This will wash in any sample residue.) Then, add a larger layer and fill the column above the silica. Be sure to add gently at first to prevent disturbing the surface of the column. Once you have a few centimeters of liquid above your column, you may pour in solvent along the sides of the column.

4. Allow the solvent to pass through the column, collecting fractions in small test tubes. Refill with mobile phase solvent as necessary. You will probably need to collect about 10 fractions.

5. Perform TLC analysis on your fractions (9:1 petroleum ether:acetone), spotting the contents of each test tube as it is filled. You can fit 5-6 fractions on a single TLC plate. Visualize the plates under UV light.

6. Use your data to decide which fractions contain pure product. Recombine these fractions in an appropriately sized pre-weighed round bottom flask. Remember that your goal is to collect a purified sample in order to characterize the product structure.

7. Remove the solvent by rotary evaporation and re-weigh the flask to determine the quantity of purified product.