Analysis of Lipid through Two-Dimensional Thin Layer Chromatography Myca Pua, Ramon Ramos, Amanda Redilas, Kimleigh Reyes,
Nathaniel Sim and Clara Tamondong
Group 9 2F Medical Technology Biochemistry Laboratory
Lipids constitute a group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins such as vitamins A, D, E, and K, monoglycerides, diglycerides, triglycerides, phospholipids, and others. In this experiment lipid was extracted from the egg yolk of a chicken egg. Three forms of lipid were extracted namely total lipid, neutral lipid and polar lipid. Then using a square chromatoplate the lipid was subjected to a two-dimensional thin layer chromatography to separate the lipid into different components. Theoretically the egg yolk should contain TAGs, cholesterol ester, cholesterol, free fatty acids and polar lipids. In the end of the experiment the chromatoplate should show the polarity of the spots based on the adsorption of the spots in the mobile phase of the two-dimensional thin layer chromatography. Proper handling of the chromatoplate and developing chamber is recommended to avoid errors in the result.
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, that’s 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 (1).
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 an equal weight of carbohydrates (2).
Composition of Chicken Egg Yolk
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, 210mg 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.
The composition (by weight) of the most prevalent fatty acids in egg yolk is typically as follows:
Unsaturated fatty acids:
Oleic acid 47 %
Linoleic acid 16 %
Palmitoleic acid 5 %
Linolenic acid 2 %
Saturated fatty acids:
Palmitic acid 23 %
Stearic acid 4 %
Myristic acid 1 %
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 (3). Two-dimensional Thin Layer Chromatography
Two-dimensional chromatography is a type of chromatographic technique in which the injected sample is separated by passing through two different separation stages. Two-dimensional chromatography can be done in column chromatography where to columns are prepared with two different solvent systems, after the eluate passes through the first column it goes to the second column. Two-dimensional chromatography can also be done in thin layer chromatography where...
References:  Bettelheim,F.A., March,J. (1990). Introduction to organic and biochemistry. Philadelphia: Saunders College.
 McKee. (2003). Biochemistry: The Molecular Basis of Life. Boston: McGraw-Hill.
 Lehninger, A.L. (2008). Legninger Principles of Biochemistry. New York: W.H. Freeman.
 Heftman, E. (1967). Chromatography. New York: Reinhold Publishing Corporation
Figure 4 Theoretical Two-dimensional TLC Chromatoplate
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