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Separation of Lipids


									                                  Separation of Lipids
        The goal of this lab is to visualize several of the various lipids found in food. Along with
various attachments (i.e. phosphate groups), these lipids can vary anywhere in length from 14-26
carbon in their fatty acid chain composition. We will use Thin Layer Chromatography (TLC) to
separate the lipids based upon their affinity for the solvent you use to dissolve them. Lipids that
are more soluble in the given solvent will move up the silica farther than lipids which are less
soluble. One of the goals of this lab is for you to determine which lipid(s) are best separated by
the given solvent and to be able to explain the chemical reason for this result. You will be
testing the solubility of 7 different lipids. The general structures of various lipids are shown

1) Working in pairs, collect 50µg/µl of seven lipids: Canola oil, coconut oil, olive oil, natural
butter, light butter, shortening, and lard.

2) In small Eppendorf tubes, dilute lipid components 1:50 in 60:30:10
chloroform:methanol:CaCl2. Note:chloroform:methanol:CaCl2 (ChMeCC) is very volatile;
the longer it is exposed to the air, the more it evaporates!! Close Eppendorf tubes
3) Get two (6) strips of chromatography paper from your instructor. Draw a straight horizontal
line, in pencil, approximately 1 cm from the lower edge of each strip of the silica.

4) Fill a glass capillary tube with diluted lipid by touching 1 end to the lipid solvent. You
should be able to see it move up the tube through capillary action.

5) Place a concentrated spot of each lipid dilution on the horizontal line on your chromatography
paper by briefly touching one end of the capillary tube to the paper. On one silica, you will have
three spots; on the other silica you will 4 spots. Some ChMeCC should escape, making a small
circle 2-4mm in diameter. Blow across this circle to dry it, then repeat until you have a dark
brown spot. The darker your spot, the easier it will be to see individual lipids as they move up
the paper. You will have 3 TLC plates with 3 spots on them, and 3 TLC plates with 4 spots.

6) Place approximately 5 mm of mobile phase solvent in the bottom of a chromatography
chamber. Do NOT add too much! If the solvent directly touches your spot of lipid it can
dissolve and move out into the solution, ruining your separation. There are 3 different mobile
phases, so that all 7 lipid varieties will run in each of the 3 mobile phases.

7) Put the lower edge of your chromatography paper into the solvent and allow the solvent to
flow up the paper. Cover the chromatography with a watch glass to reduce evaporation and
allow the mobile phase to move up the chromatography paper until it reaches the top.

8) Remove the paper from the solvent and air dry it.

9) Stain silica in Primuline for 30-35 seconds. Air dry.

10) Next, under imaging scanners in room 332, look at silica(s) in Quantity One under TransUV

Canola oil,
Coconut oil
Olive oil
Natural butter
Light butter
60:30:10 chloroform:methanol:CaCl2 (v/v/v)
0.5ml eppindorf tubes
Glass capillary tubes (pulled from pasteur pipettes)
1 mL pipettes
Analytical Balance
Chromatography paper
Chromatography Chamber

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