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

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.

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 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 orexpanded 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. 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

through 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.



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 run-

Extraction of Total Lipids from Chicken Egg Yolk. Lipids are soluble in organic solvents, but sparingly soluble or insoluble in water. The existing procedures for the extraction of lipids from source material usually involve selective solvent extraction and the starting material may be subjected to drying prior to extraction. Solubility of lipids is an important criterion for their extraction from source material and depends heavily on the type of lipid present, and the proportion of nonpolar (principally triacylglycerols) and polar lipids (mainly phospholipids and glycolipids) in the sample; therefore, several solvent systems might be considered, depending on the type of sample and its component. The solvents of choice are usually hexane, chloroform, methanol or chloroform, methanol or water. The simple lipids include waxes, fats, and oils. These compounds are structurally similar to each other because they consist of alcohols combined with long organic acids known as fatty acids. Waxes are constructed of a single molecule each of alcohol and acid while fats and oils contain three fatty acid molecules for each alcohol molecule. Fats are distinguished from oils in that the former are solids and the latter, liquids. Compound lipids consist of a simple lipid and some other group, such as a phosphoric acid fragment or a nitrogen-containing alcohol. Members of this family have many important functions in biological systems. For example, one group of compound lipids, the glycolipids, occur in the membranes of brain and nerve cells. Because the compound lipids are so complex and because they occur in such small concentrations, little has been known about them until recently. One of the best known compound lipids is lecithin. Lipoprotiens are organic compounds composed of both protein and a lipid. There are at least four groups of lipoproteins present in plasma: Highdensity lipoproteins (HDL), low-density

lipoproteins (LDL), very low density lipoproteins (VLDL), and chlyomicrons. The different densities refer to the relative amounts of lipid and protein. The higher the density, the higher the protein to lipid ratio. LDLs transport cholesterol to cells and deposit excess cholesterol in the blood vessels, which increases the risk of arteriosclerosis. HDLs, however, transport cholesterol from the tissues to the liver where it is excreted, lowering the risk of arteriosclerosis. A high HDL to total cholesterol ratio is the best indication of decreased risk of arteriosclerosis. HDL levels vary from person to person and can be influenced by such things as heredity, sex, age, and physical activity. Smoking and obesity have been shown to decrease plasma HDL levels. The composition of chicken egg 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. All of the fat soluble vitamins, (A, D, E and K) are found in the egg yolk. 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 yolks are one of the few foods naturally containing vitamin D. The yellow color is caused by lutein and zeaxanthin, which are yellow or orange carotenoids known as xanthophylls. Column Chromatography of Lipids. The table below (Figure1) gives us the results obtained in this experiment. With the use of the collected lipid extract from chicken egg yolk, the lipids present in the crude extract was analyzed and the first eluate was triacylglyceride, second eluate was cholesterol, and the third eluate is the lecithin. Eluates 1st eluate 2nd eluate 3rd eluate Components Triacylglycerol Cholesterol Lecithin

chromatography) and the more polar the lipid, the more strongly is it adsorbed. Thus, the lipids are eluted by increasingly polar solvents. This technique has a low resolution when used at low pressure (Solid Phase Extraction or SPE) but has a high resolution (high performance) when run at high pressure using a stationary phase made of fine particles (HPLC). The former is restricted to the fractionation of complex mixtures into two or three less complex ones, the later being adopted to analyze and quantify purified fractions. Lecithin is the most polar among the three eluates, next is cholesterol and last is triacylglycerol. The first eluate was triacylglycerol or triglyceride. The eluent was 5 mL 9:1 mixture of petroleum ether:ethyl ether. Triacylglycerols are the main components of animal and plant lipids. They are an ester of three fatty acids and glycerol. They are the most concentrated source of energy in the human body and are stored in subcutaneous fat deposits where they contribute to insulation. Triglycerides have lower densities than water (they float on water), and at normal room temperatures may be solid or liquid. When solid, they are called "fats" or "butters" and when liquid they are called "oils". Independent studies of biosynthesis of fatty acid and glycerol components of glycerolipids exhibited that 3H-leucine was mainly consumed in synthesis of glycerol moiety of phospholipids and triacylglycerols, whereas 14C-acetate was utilized in synthesis of fatty acids. Ethanol activated most distinctly the synthesis of glycerol moiety as compared with the synthesis of triacylglycerol fatty acids. Ethanol activated more effectively esterification of fatty acids with formation of triacylglycerols as compared with phospholipids. Incorporation of the label into glycerol molecule occurred in response to activation of glycero-glyconeogenesis by ethanol. The image below (Figure2) gives us the structure of triacylglyceride. The second eluate was cholesterol. The eluent was 5 mL 5% methanol in dichloromethane. Cholesterol is a chemical compound that is naturally produced by the body and is a combination of lipid (fat) and steroid. Cholesterol is a building block for cell membranes and for hormones like estrogen and testosterone. About 80% of the body's cholesterol is produced by the liver, while the rest comes from our diet. Cholesterol is only one of several lipids (fats) circulating in our blood stream. Its components, Triglycerides are an additional form of fat (3 fatty

Figure1. Components of Eluates Chromatography of lipids using a glass column filled with a suitable material is a common and useful method for fractionation of lipid classes either on an analytical or a semi-preparative scale. The retention results in a variety of mechanisms including hydrogen bonding, Van der Waals' forces and also ionic bonding. The solid phase is relatively polar (normal

acids plus glycerol) circulating in the blood. Cholesterol and Triglycerides cannot dissolve in water due to being lipids, or fats. Because our blood is comprised primarily of water, for Cholesterol and Triglycerides to circulate through your blood, the Cholesterol and Triglycerides must be carried by protein packages called Apoproteins. The third eluate was lecithin. The eluent was 5 mL dichloromethane:methanol:water (1:3:1). Lecithin is a lipid that consists mostly of choline, but also includes inositol, phosphorus, and linoleic acid. Lecithin helps to prevent arteriosclerosis, protects against cardiovascular disease, improves brain function, helps keep the liver and kidneys healthy, aids in thiamin and vitamin A absorption, and can even help to repair liver damage caused by alcoholism, this nutrient is essential to every living cell in the human body. One of the various functions of lecithin is to keep cholesterol in line. Its ability to emulsify oils and hold them in solution plays a major role in preventing gall stone formation. Together with bile and bile salts, it comprises the three major constituents of bile. Bile is mostly made up of fats, which lecithin keeps in liquid form in order to prevent gall stones from forming. On the other hand, cholesterol holds a delicate balance with the bile salts. If the balance is tipped on either side, the result could stone formation. By keeping cholesterol in check, lecithin helps prevent stone formation. As a component of the enzyme lecithin cholesterol acyl tranferase, the compound is said to help in the metabolism of cholesterol to its by products. As mentioned earlier, this substance is also called phosphatidylcholine and is an excellent source of choline. Much of the medical benefits of lecithin, particularly on high cholesterol-related conditions have been attributed to the presence of choline. The image below (Figure4) gives us the structure of lecithin.

We have concluded that the lipids extracted from the chicken egg yolk are separated based on its differences in solubility. There are factors affecting lipid solubility like chemical nature of the molecule, atomic or molecular formula weight, valence or charge or sphere of hydration, charge density and sphere of hydration. Also, in analyzing the lipids present in the crude extract using column chromatography, it is necessary to first isolate them quantitatively from nonlipid components. Extraction of lipids from source materials, such as food, animal and plant tissues, or microorganism, should be carried out in a manner that avoids changes in the lipids or leads to the formation of artifacts it has eluates and each was differentiated by its components.


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