Project 4 Procedure

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

Week 1

Reaction A. In a 25 mL round bottom flask, 2,4-pentanedione (10 mmol) was added to a mixture of glycine (10 mmol) and potassium hydroxide (10 mmol) in methanol (7 mL). The mixture was refluxed for 30 minutes. (Note: Lightly grease the joint between the flask and the condenser. See Reminder note #2 below.) After the reflux period, the flask was cooled briefly in an ice-water bath. The mixture was then poured into ice-cold 1:1 hexane:acetone (40 mL). The resulting solid was collected by vacuum filtration, and allowed to dry under vacuum for 10 minutes. The mass was recorded, then the appropriate quantity of solid was used for Reaction B. An IR spectrum and ¹H/¹³C NMR spectra (solvent: d6-DMSO) were collected from the remaining solid. (Note: The product is only moderately soluble in DMSO. To prepare your sample, add a little solid to a test tube. After you add the DMSO, use a pipet to gently mix the contents and dissolve most of the solid. If your sample does not fully dissolve, you can remove the excess solid by filtering the solution through a Kimwipe into your NMR tube).

Reaction B. In a 250 mL round bottom flask, a mixture of the Reaction A product (5 mmol), triethylamine (35 mmol), and acetic anhydride (159 mmol) was refluxed for 20 minutes. (Note: Use a larger stir bar to keep your reaction well-mixed. Upon heating, vigorous bubbling should be observed for several minutes. This will subside and the solid will dissolve completely). After the reflux period, the mixture was allowed to cool for 5 minutes whereupon a solid formed in the flask. Distilled water (150 mL) was added and the mixture was stirred vigorously at room temperature for 20 minutes. The reaction mixture was transferred to a separatory funnel and extracted with ethyl acetate (2 × 40 mL). The combined organic layers were washed sequentially with water, saturated sodium bicarbonate, and brine. The solution was dried over sodium sulfate and gravity filtered. The solvent was removed by rotary evaporation, and the resulting oil was saved in a capped test tube for purification during the next lab meeting.

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.

3. Remember that the Reaction A NMR sample must be dissolved in d6-DMSO rather than the usual CDCl3. You should check here to see where to expect the DMSO peak in your NMR spectrum.

4. When you perform the extraction for Reaction B, some of the product may stick to the glass of the round bottom flask. Use the ethyl acetate to wash residual product from the flask into the separatory funnel before proceeding with your extraction!

Week 2

Isolate the product pyrrole (Rf ≅ 0.27) by column chromatography using a large diameter column, ~30 mL silica gel, and a 95:5 hexane:acetone mobile phase. If the oil is too viscous to transfer to the column, you dilute it with a small amount of acetone. Collect fractions in 13 × 100 mm test tubes. Recombine only fractions containing the product and remove the solvent by rotary evaporation. After the solvent has evaporated, allow the flask to remain on the rotary evaporator for 15 minutes. Obtain GC-MS, IR, and ¹H/¹³C NMR (CDCl3) data for the product.

Reminders & Hints

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

2. To refresh your memory on column chromatography, review the technique video below. The video uses slightly different materials than required for this experiment, but the process is the same.  For this experiment, your silica should be about 5.5 cm tall (roughly 1/3 the height of the plastic column). In addition, a step-by-step example procedure is given below.

Column Chromatography Step-by-Step

1. Build the chromatography column:

a. In a 150 mL beaker, mix about 30 mL of silica gel with 95:5 hexane: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 (~0.1 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 (95:5 hexane:acetone), spotting the contents of each test tube as it is filled. You can fit about 5 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.