Fractionation Of Lipids

Words of Caution:
You will be working with very volatile, flammable organic solvents. Therefore, work in the fume hood, do not work near any open flames and be sure to dispose of the excess solvents in the excess SOLVENT container.
Use tightly fitting stoppers to seal your tubes to prevent evaporation of the solvent. Also, storing your tubes at -20 *C (the freezer) will also minimize evaporation.
Lipids are highly susceptible to oxidation and polymerization. To avoid this, put a small amount of nitrogen gas over the top of your tubes before sealing and then store the samples in the dark. (The freezer is a dark place.)

Week 2: Column Fractionation of Lipids

note: ether = ethyl ether = diethyl ether
1. Calculate the volume of the "total lipids" from the gravimetric analysis. Use the weight of the "total lipids" on the aluminum weighing boat and back calculate the total amount of lipids in your screw cap tubes, then determine how much of this volume will contain 70- 80 mg.
2. Make up approximately 30 mL of ether:methanol (100:1, v/v), 100 mL of ether + 1 mL methanol—but adjust the ratios for a total volume of of 30 mL. Store this in a stoppered erlenmeyer flask.
3. Evaporate this amount of lipids to dryness under nitrogen gas and redissolve these lipids in 2 mL of a mixture of ether:methanol 100:1 (v/v).
4. Clamp a syringe barrel in the vertical position, with the tip down.
5. Rinse the syringe with acetone and then with ether:methanol (100:1, v/v), and allow the syringe to dry.
6. Attach the syringe to the plastic column that contains silica as a packing material using an adapter. Note: These syringe-column adaptors are not disposable. You must return them to the stockroom, or we will come after you.
7. Set up a holder for a test tube that will fit under the silica column.
8. Label a set of test tubes:

Neutral fraction
Glycolipid fraction
Polar fraction

9. Make up the following solutions of organic solvents. Put each solution in a screw cap tube.

Solutions in tubes:
2 mL of "total lipids" (from #3 above)
2 mL of ether:methanol (100:1, v/v)
10 mL ether:methanol (100:1, v/v)
8 mL acetone
8 mL acethone: acetic acid (100:1, v/v). This is 100 mL of acetone and 1 mL of glacial acetic acid, but adjust the volumes for a total of 10 mL and then use 8 mL of that.
5 mL ether:methanol (1:1, v/v)
5 mL of methanol

Note: Do not let the silica column run dry, once you have started adding solutions to your column, you are committed to finishing the job. Make sure you have all the solutions and test tubes that you need.

10. Load the "total lipids" on the column, then add 2 mL of ether:methanol (100:1, v/v) (tube 2 from #9 above) to wash out the "total lipids" tube and add this to the column. Collect the the column effluent in the tube labelled "Neutral Lipids".
11. Add 10 mL ether:methanol (100:1, v/v) and continue to collect the effluent in the tube labelled "Neutral fraction".
12. Add 8 mL acetone and after 1 mL of the acetone has entered the column, begin collecting the effluent in the tube labelled "Glycolipid fraction" .
13. Add 8 mL acethone: acetic acid (100:1, v/v) and continue collecting the effluent in the the "Glycolipid fraction" tube.
14. Add 5 mL ether:methanol (1:1, v/v) and begin collecting the effluent in the tube labelled "Polar fraction".
15. Add 5 mL of methanol and continue collecting the effluent in the "Polar fraction" tube.
16. Using a graduated cylinder, bring the volumes of each fraction up to 20 mL with ether. Transfer 5 mL of each fraction to a pre-weighed aluminum weighing boat for gravimetric analysis. Remember to use forceps and weighing papers to handle the aluminum weighing boats. Cover each boat with a watch glass and allow them to evaporate in the hood overnight.
17. Transfer the remainder of each fraction to a screw cap tube and dry each fraction under nitrogen in a warm water bath.
18. Redissolve the the neutral fraction in 5 mL ether:methanol (100:1, v/v).
19. Redissolve the polar fraction in 5 mL of ether:methanol (1:3, v/v)
20. Put a layer of nitrogen over each fraction, place the screw cap lids on each tube and store them in the freezer.

For the gravimetric analysis:

Report the yeild of each fraction as mg fraction/g of egg yolk
Calculate the percent of total lipids in an egg yolk
Calculate the percent recovery from the silica column.

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