TLC Exploration of Spearmint Oil
1. Prepare 3 TLC chambers, one for each of the following mobile phases: 9:1 Hexane:Ethyl Acetate; Hexane; Ethyl Acetate.
2. Prepare 3 TLC plates. On each plate, spot four samples: spearmint oil, limonene, carvone, and dihydrocarveol.
3. Allow each TLC plate to develop in one of the three chambers.
NOTE: Start preparing your chromatography column while you wait for the TLC plates to develop.
4. After each TLC plate is developed, remove it from the TLC chamber and visualize it under the UV light and using the anisaldehyde stain. (Your instructor will demonstrate use of the stain.) Be sure to circle any spots you see on the plates.
5. Decide which solvent gave the best separation and calculate the Rf values for the spots on that plate.
Column Chromatography of Spearmint Oil
1. Build the chromatography column:
a. In a 150 mL beaker, mix about 50 mL of silica gel with 9:1 Hexane:Ethyl Acetate until you have a slurry (thin milkshake consistency).
b. Clamp the column in place, place an Erlenmeyer flask beneath it (to collect solvent that passes through), and add the slurry of silica. The height of the silica column should be 1/3-1/2 that of the plastic column. Repeat as necessary to increase the height of your column if you did not add enough silica the first time.
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 thin layer of sand on top of the silica. Wash down any sand from the walls of the column with solvent.
f. Use a Pasteur pipet to add a few milliliters of solvent to the top of the column and allow drain until it reaches the top of the column.
2. Using a Pasteur pipet, add ~1.5 mL of spearmint oil to the column using the same swirling/gentle addition method described above. NOTE: Make sure you are using pure spearmint oil, not the diluted TLC samples. 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.
3. Add the mobile phase to the top of the column: Start will just a thin layer and allow that pass into the column. Then, add a larger layer and fill the space above the column. Be sure to add gently at first to prevent disturbing the surface of the column. Once you have a few inches of liquid above your column, you may pour in solvent to fill the reservoir at the top 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 at least 24 fractions.
5. Perform TLC analysis on your fractions, using the best solvent system and spotting each test tube as it is filled. You can fit 5-6 fractions on a single TLC plate. Visualize the plates under UV and with permanganate stain.
6. Use your data to decide which fractions contain pure carvone. Recombine these fractions in an appropriately sized pre-weighed round bottom flask.
7. Remove the solvent by rotary evaporation and re-weigh the flask to determine the quantity of carvone you isolated.
8. Fully characterize the carvone by GC-MS, IR, and NMR.
• Your instructor or TA will demonstrate how to prepare an NMR sample.
• The GC-MS sample is prepared by dissolving 1 drop of your sample into ~ 2 mL ethyl acetate.
9. Save your isolated carvone in a labeled test tube capped with a rubber septum.
Video Preview
The video below demonstrates how to perform column chromatography using the materials available in our laboratory.