Extraction Of Lipids From Egg Yolks

Isolation of Lipids from Egg Yolks

Grinding the tissue with sand and methanol:

Separate an egg yolk from the white and then divide the yolk into four portions. Weigh (to the nearest 0.1 g) one portion and then transfer it to a mortar and add an equal (eyeball) amount of acid washed sand, 5 mL of methanol and grind to a smooth paste. Transfer your paste to two 16 x 125mm centrifuge tubes (using another 5 mL of methanol to wash out your mortar) and heat to 60*C in a water bath. After 3 minutes, remove the centrifuge tube from the hot water bath and allow it to cool to room temperature.

Extracting with diethyl ether:

Transfer the egg yolk paste back to the mortar, using 5 mL of diethyl ether to wash out your centrifuge tube, and grind again. (This is a good chance to relieve any frustrations you may have in life—just grind them away!) Return this suspension to your 16 x 125 mm centrifuge tubes with another 5 mL of diethyl ether. Cover the tubes with aluminum foil and centrifuge in a table top centrifuge for 5 minutes. Be sure the centrifuge is in the hood and that you have the opposite tubes balanced.
Decant the supernatant into a 50 mL Erlenmeyer task. Mix up a 20 mL solution that is methanol:diethylether (1:1). Add 5 ml of this solution to the pellet in your 16 x 125 mm centrifuge tubes, cover the tube with aluminum foil, heat cautiously (60*C water bath) for three minutes and then centrifuge for 5 minutes. Again, decant the supernatant into your 50 mL Erlenmeyer flask. Repeat this extraction two more times, adding the supernatants to the 50 mL Erlenmeyer flask.

Filter the diethyl ether extracts:

Place 40 mL of an isotonic (0.9% NaCl w/v = 0.9 grams of NaCl in 100 mL of distilled water) saline solution in a 100 mL graduated cylilnder. Place a funnel with a fluted filter paper (Whatman #2—read the label on the box of filters to make sure.) on top of the graduated cylinder and carefully pour the combined extracts from your 50 mL Erlenmeyer flask into the filter paper. Rinse the Erlenmeyer flask with 10 mL of diethyl ether and add the ether to the filtering apparatus.

Removal of non lipid water soluble contaminants:

Mix the contents of the graduated cylinder with a clean glass stirring rod. Then, allow the mixture to separate into two layers. Transfer the clear diethyl ether layer to a clean 25 mL graduated cylinder with a Pasteur pipet. Add 5 mL of diethyl ether to the 100 mL graduated cylinder, mix, allow to separate and transfer the diethyl ether layer to the 25 mL graduated cylinder. Repeat this addition of 5 mL of diethyl ether twice more, adding the diethyl ether layers to the 25 mL graduated cylinder. Then, adjust the volume to 25 mL by either adding diethyl ether, or evaporating the solvent with a flow of nitrogen gas. This is your total lipids extract.
Begin Gravimetric Analysis:

Label an aluminum weighing boat #1,

and place a piece of weighing paper on the balance and tare the balance. Then, using forceps, place the boat on weighing paper and then weigh the boat to the nearest 0.001g. Repeat this weighing procedure two more times. Pipet 5 mL from the 25 mL graduated cylinder onto the preweighed aluminum weighing boat. Cover the boat (that is still on the weighing paper) with a watch glass and allow it to dry in the hood overnight. (It is important not to touch the boat—your fingers will leave grease that will increase the weight.) This will be the amount of your total lipids.

Before leaving:

Transfer the remaining 20 mL of lipid extract to two 16 x 125 mm centrifuge tubes with screw caps. Cover the top of the tube with a layer of nitrogen gas. Label the tubes "Total Lipids" and put your name and the date on it before storing it in the freezer.

Unless otherwise stated, the content of this page is licensed under Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License