Purification of a Total Lipid Extract with Column Chromatography

1. Setup and Preparation of Materials

1. Obtain a total lipid extract (TLE) using a solvent extraction method (Sonication, Soxhlet, or Accelerated Solvent Extraction (ASE)).
2. Gather the following: combusted borosilicate glass pipettes and bulbs, silica gel, hexane, dichloromethane (DCM), and methanol.
   1. These materials can be purchased from any chemical retailer. The reagents should be pure and free from hydrocarbons.
3. Also obtain combusted glass wool and 4-mL borosilicate glass vials.
4. Make sure to have a means of supporting the column and the collection vials during the procedure. For example, a stand with clamps and a vile rack. Many labs have engineered custom-made racks that hold several columns and collection vials (Figure 2). This allows many columns to be run at the same time.

2. Methods

1. Start with the dry sample in a 4-mL vial. If the sample weighs more than ~ 10 mg when dry, it may need to be split before performing the next steps as the silica gel can only react with a finite mass of organic matter.
2. Suspend the sample in a small amount of hexane. This is the first and the least polar of the three solvents used in this experiment.
3. If there is sample stuck to the inside of the vial, sonicate the samples for 5 min.
4. Load a small amount of glass wool into the top of a pipette using a set of clean tweezers. Gently push the glass wool to the bottom of the pipette, using another pipette, to form a plug.
5. Carefully transfer silica gel into the pipette until it is approximately half full.
6. Place a 4-mL waste collection vial under the column.
7. Soak the silica gel in the pipette with 3 volumes of hexane. This conditions the column, removes air bubbles, and rinses any impurities off the silica gel.
8. Once the final wash is done, remove the waste collection vial and replace it with a vial to collect the apolar fraction.
9. Carefully transfer the entire sample in hexane onto the column using a pipette. Rinse the sample vial two more times with small volumes of hexane, and transfer to the column. Allow the hexane the sample was transferred in to completely soak into the silica gel. At no time during the procedure should the silica gel dry out.
10. Continue adding hexane to the top of the sample until the collection vial below the column is nearly full (~4 mL).
    1. Allow all of the hexane to enter the silica gel before starting with the next solvent.
11. Place the mid-polarity collection vial under the column.
12. Add DCM to the top of the column until the collection vial is nearly full. Again, allow all of the DCM to enter the collection vial before starting the next solvent.
13. Place the polar collection vial under the column.
14. Add methanol to the top of the column until the collection vile is nearly full.