Words of Caution:
You will be working with very volatile, flammable organic solvents. Therefore,
Use tightly fitting stoppers to seal your tubes to prevent evaporation of the solvent.
Also, storing your tubes at -20 *C (the freezer in S208) 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 in S208 is a dark place.)
Be sure to consult the Properties of Organic Solvents Table at the Chemistry Stockroom
Window.
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:
8. Make up the following solutions of organic solvents. Put each solution in a screw
cap tube.
Solutions in tubes:
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.
9. Load the "total lipids" on the column, then add 2 mL of ether:methanol
(100:1, v/v) (tube 2 from #8 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".
10. Add 10 mL ether:methanol (100:1, v/v) and continue to collect the effluent in the tube
labelled "Neutral fraction".
11. 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" .
12. Add 8 mL acethone: acetic acid (100:1, v/v) and continue collecting the effluent in
the the "Glycolipid fraction" tube.
13. Add 5 mL ether:methanol (1:1, v/v) and begin collecting the effluent in the tube
labelled "Polar fraction".
14. Add 5 mL of methanol and continue collecting the effluent in the "Polar
fraction" tube.
15. 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.
16. Transfer the remainder of each fraction to a screw cap tube and dry each fraction
under nitrogen in a warm water bath.
17. Redissolve the the neutral fraction in 5 mL ether:methanol (100:1, v/v).
18. Redissolve the polar fraction in 5 mL of ether:methanol (1:3, v/v)
19. Put a layer of nitrogen over each fraction, place the screw cap lids on each tube and
store them in the freezer.
Back to the Lipids page
Forward to the instructions for part 3.
Last updated by K. Stone on April 24, 2001