EXTRACTION OF TOTAL LIPIDS FROM CHICKEN EGG YOLK AND COLUMN CHROMATOGRAPHY

Christine Umali, Jon Lester Uy, Chloe Valena, Jose Maria Veloso, Norjem Villanueva and Jaquelyn Wodi Group 9 2A Medical Technology BioChemistry Laboratory

ABSTRACT
The experiment was about extraction of total lipids from chicken egg yolk and column chromatography of. Total lipids were extracted from the chicken egg yolk using 1M NaCl, isopropyl alcohol and petroleum ether. The mixture was left standing for five minutes. The lower layer was collected and subjected into column chromatography. The column used for column chromatography was packed with slurry of 0.5 g silica gel in 4 ml of petroleum ether with tapered end glass wool. For this experiment, the column was washed three times using different eluents. The first eluent used was of 9:1 mixture of petroleum ether and ethyl ether, the second eluent was 5 ml 5% methanol in dichloromethane and the last eluent was 5ml CH2Cl2: CH3OH: H2O (1:3:1). Eluates for each eluent introduced into the column were collected in separate test tubes. Collected eluates were subjected for the qualitative tests for lipids. 10 drops of eluates were subjected for each qualitative test.

INTRODUCTION
Lipids are substances found in living organisms that are insoluble in water but soluble in non polar solvents and solvents of low polarity. This lack of solubility in water is an important property because our body chemistry is so firmly based on water. Most body constituents including carbohydrates which are soluble in water. But the body also needs insoluble compounds for many purposes, including the separation of compartments containing aqueous solutions from each other, thats where lipids come in. The water-insolubility of lipids is due to the fact that the polar groups they contain are much smaller than their alkane-like (nonpolar) portions. These nonpolar portions provide the water-repellent, or hydrophobic, property. An important use for lipids, especially in animals, is storage of energy. Plant store energy in form of starch. Animals including humans find it more economical to use lipids (fats) instead. Although our bodies do store some carbohydrates in the form of glycogen for quick energy when we need it, energy stored in the form of fats is much more important. The reason is simply that the burning of fats produces more than twice as much energy as burning of an equal weight of carbohydrates. The yolk makes up about 33% of the liquid weight of the egg; it contains approximately 60 calories, three times the caloric content of the egg white. One large egg (50 grams in weight, 17 gram yolk) contains approximately: 2.7g protein, 210 mg cholesterol, 0.61g carbohydrates and 4.51g total fat. (USDA National Nutrient Database) All of the fat soluble vitamins, (A, D, E and K) are found in the egg yolk. Egg yolks are one of the few foods naturally containing vitamin D. Egg yolk is a source of lecithin, an emulsifier and surfactant. The yellow color is caused by

lutein and zeaxanthin, which are yellow or orange carotenoids known as xanthophylls. Column chromatography is one of the most useful methods for the separation and purification of both solids and liquids when carrying out small-scale experiments. Column chromatography is another solid-liquid technique in which the two phases are a solid (stationary phase) and a liquid (moving phase). The theory of column chromatography is analogous to that of thin-layer chromatography. The most common adsorbents - silica gel and alumina - are the same ones used in TLC. The sample is dissolved in a small quantity of solvent (the eluent) and applied to the top of the column. The eluent, instead of rising by capillary action up a thin layer, flows down through the column filled with the adsorbent. Just as in TLC, there is an equilibrium established between the solute adsorbed on the silica gel or alumina and the eluting solvent flowing down through the column. The objectives of the experiment are as follows: (1) to extract total lipids from chicken egg yolk, (2) to analyze the lipids present in the crude extract using column chromatography The stationary phase or adsorbent in column chromatography is a solid. The most common stationary phase for column chromatography is silica gel, followed by alumina. Cellulose powder has often been used in the past. Also possible are ion exchange chromatography,reversed- phase chromatography (RP), affinity chromatography or expanded bed adsorption (EBA). The stationary phases are usually finely ground powders or gels and/or are microporous for an increased surface, though in EBA a fluidized bed is used. The mobile phase or eluent is either a pure solvent or a mixture of different solvents. It is chosen so that the retention factor value of the compound of interest is roughly around 0.2 - 0.3

in order to minimize the time and the amount of eluent to run the chromatography. The eluent has also been chosen so that the different compounds can be separated effectively. The eluent is optimized in small scale pretests, often using thin layer chromatography (TLC) with the same stationary phase.

REFERENCES
Bettelheim,F.A., March,J. (1990). Introduction to organic and biochemistry. Philadelphia: Saunders College. Heftman, E. (1967). Chromatography. New York: Reinhold Publishing Corporation Lehninger, A.L. (2008). Legninger Principles of Biochemistry. New York: W.H. Freeman. McKee. (2003). Biochemistry: The Molecular Basis of Life. Boston: McGraw-Hill. WEBSITES: http://faculty.mansfield.edu/bganong/bioch emistry/liptlc2.htm Retrieved: March 7, 2012 http://orgchem.colorado.edu/hndbksupport/ colchrom/colchrom.html Retrieved: March 7, 2012 http://lipidlibrary.aocs.org/Lipids/whatlip/in dex.htm#def Retrieved: March 7, 2012

EXPERIMENTAL
A. Compounds Tested The following were used for the Extraction of total lipids from chicken egg yolk: Chicken egg yolk, 5 volumes of 1M NaCl, 3ml isopropyl alcohol and 2ml petroleum ether. The following were used for the column chromatography of lipids: extracted total lipids from chicken egg yolk, 0.5 g silica gel, 4ml of petroleum ether, 5ml of 9:1 mixture of petroleum ether and ethyl ether, 5 ml 5% methanol in dichloromethane, and 5ml CH2Cl2: CH3OH: H2O (1:3:1). B. Procedure 1. Extraction of Total Lipids from Chicken Egg Yolk The egg yolk was separated from then chicken egg and its volume was determined. It was then diluted with 5 volumes of 1M NaCl. After dilution, 2ml of the diluted egg yolk was mixed with 3ml isopropyl alcohol in a separate clean test tube. Petroleum ether with a volume of 2 ml was then added. It was covered with rubber stopper and was ensured to be well mixed. The mixture was then allowed to stand for 5 minutes until two layers were formed. After 5 minutes, the lower layer was collected and was transferred to another clean test tube. Two-dimensional thin layer chromatography (TLC) and column chromatography (CC) was performed using the lower layer. 2. Column Chromatography of Lipids Small column was prepared by pouring a slurry of 0.5 g silica gel in 4ml of petroleum ether into the glass column (Pasteur pipette) with a tapered end plugged with glass wool. The lipid extract from chicken egg yolk with a volume of 1 ml was then introduced into the column, saving the runthrough in a clean test tube. The column was washed with 5ml 9:1 mixture of petroleum ether and ethyl ether, collecting the eluate in the same tube as the runthrough. The column was again washed with the second eluent (5 ml 5% methanol in dichloromethane) the eluate was then collected in another clean test tube. The column was washed with the last eluent, 5ml CH2Cl2: CH3OH: H2O (1:3:1) and eluate was collected in another test tube. The different eluates culled were saved for qualitative analysis.

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Retrieved: March 7, 2012