EXTRACTION OF TOTAL LIPIDS FROM CHICKEN EGG YOLK AND QUALITATIVE TESTS FOR LIPIDS Camaclang, Rosa Magenta

Comia Jasmin Contreras Charles De Leon Angelica Luz *Dela Cruz Kizer Group 2 – 2BPH ABSTRACT
Lipid molecules include fats, waxes, and fat-soluble vitamins such as A, D, E and K. A column chromatographic procedure utilizing silica gel is described for separating lipid components of serum and lipoproteins into individual fractions containing hydrocarbons, cholesterol esters, triglycerides, cholesterol, free fatty acids and phospholipids. Egg-yolk lecithin has phospholipid classes and compositions that differ from soybean lecithin and may have unique functional properties. This experiment determined the components of each eluents. Lipids were based upon their polarity using column chromatography. The extracted lipids from chicken egg yolk were used in the column chromatography. The eluents used were 9:1 mixture of petroleum ether:ethyl ether, 5% methanol in dichloromethane and dichloromethane:methanol:water (1:3:1). The results obtained were analyzed and it showed that the lipids are eluted by increasingly polar solvents. The lipids present in the crude extract were triacylglycerol, cholesterol and lecithin. The aim of this experiment is to understand and to determine the amounts of lipid components in chicken egg yolk. In the end of this experiment we had founded which lipid component in chicken egg yolk is the most polar among all using column chromatography.

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, 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 non polar portions provide the water-repellent, or hydrophobic, property. An important use for lipids, especially in animals, is storage of energy. Plant stores 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 energy than burning of an equal weight of carbohydrates. In the experiment, the lipids present in chicken egg yolk were isolated. The egg yolk makes

up about 33% of the liquid weight of the egg and it contains approximately 60 calories, three times the caloric content of the egg white. All of the fatsoluble vitamins (A, D, E, and K) are found in the egg yolk. Egg yolk is one of the few foods naturally containing vitamin D. Egg yolk is a source of lecithin. Its yellow color is due to lutein and zeaxanthin, which are the yellow or orange carotenoids also known as xanthophylls. The general approach is to extract total lipids from egg yolk using methanol and chloroform, then separate the lipid fractions, triglyceride, cholesterol, and phospholipids, by chromatography on silica gel. This experiment will investigate the properties of lipids present in chicken egg yolks, the lipid components present in the crude extract using column chromatography of the extracted lipids from chicken egg yolk. 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 (3) to identify lipids present in each of the fractions using qualitative tests and, (4) to determine the degree of unsaturation of lipids by bromine test.

METHODOLOGY A. Extraction of Total Lipids from Chicken Egg Yolk
The materials we have used were test tubes, beaker, stirring rod, Pasteur pipette, hot plate, iron stand and iron clamp. We started the procedures by extracting total lipids from chicken egg yolk. We added an equal amount of ethanol to the egg yolk to increase the polarity of the organic solvent, and mixed it to dehydrate and partially extract the polar lipids. We added hexane and then mixed it again and we had let it stood for 5 minutes, until two layers were formed, the fractions of polar and neutral lipids. We removed the upper polar fraction and added an equal amount of acetone to further precipitate the polar lipids from residual neutral ones, especially the cholesterol. . When the

drops of the eluate in a test tube. The test tube must be warmed before observing any changes in the appearance of the sample. IV. Test for Cholesterol (Liebermann-Burchard Test) Ten drops of eluate was placed in a test tube and 0.25mL of dichloromethane was added. Six drops of acetic anhydride and 2 drops of concentrated H2SO4 was added and mixed with the sample. A greenish color produced after a few minutes indicated the presence of cholesterol. V. Test for Lipid Unsaturation with I2 This portion of the experiment was slightly modified. Supposedly, Br2 will be used instead of I2. But the principle involved here is the same, the only difference was the reagent used. Ten drops of each eluate was placed in different test tubes. Aside from the three eluates, 10 drops of Canola oil was also used. I2 was added dropwise in every test tube until a desired color was achieved. In this case, it should be light yellow or colorless. The color of each sample should be the same, if not; the colors should be close enough to each other

upper layer was collected, it was transferred into one clean test tube and it was used in ThinLayer Chromatography and Column Chromatography. The eluates were used for the qualitative tests for lipids B. Qualitative Tests for Lipids

I. Test for Ester EtOH: 1-BuOH (3:1) with a volume of 0.5 ml was introduced into the 10 drops of eluate. 2 drops each of 2M hydroxylamine Hydrochloride and 3M NaOH was sequentially added and was mixed well. The samples were allowed to stand for 5 minutes. 2 drops of 6M HCl was added with 1 drop of 5% FeCl3. 6 H2O in 0.1M HCl and II. Test for Glycerol (Acrolein Test) A pinch amount of KHSO4 was added to 10 drops of the eluate in the test tube. Test tube was heated in a boiling water bath and odor produced was noted. Burned fat odor was observed for positive test results. III. Test for Glycerol (Kraut’s Test) Unfortunately the reagent was not available so this test was not performed. But the steps in performing this specific test were also discussed. First, 3mL of Kraut’s reagent is to be added to 10

RESULTS AND DISCUSSIONS Table 1: Actual Results of Qualitative Tests for Lipids Chemical 1st eluate 2nd eluate 3rd eluate Ester Yellow solution Yellow solution Burgundy color Glycerol LiebermannBurchard Test No odor Blue green solution No odor Green solution Burnt fat Colorless odor solution

Table 1 shows the actual results for each qualitative performed in lipids. The principles or mechanisms behind each qualitative test are as follows: Test for Ester Esterification is the general name for a chemical reaction in which two reactants (typically an alcohol and an acid) form an ester as the reaction product.

Esters are common in organic chemistry and biological materials, and often have a characteristic pleasant, fruity odor. This leads to their extensive use in the fragrance and flavor industry. Ester bonds are also found in many polymers. Esterification is a reversible reaction. Hydrolysis—literally "water splitting"—involves adding water and a catalyst (commonly NaOH) to an ester to get the sodium salt of the carboxylic acid and alcohol. As a result of this reversibility, many esterification reactions are equilibrium reactions and therefore need to be driven to completion according to Le Chatelier's principle. Esterifications are among the simplest and most often performed organic transformations. The most common esterification processes involve nucleophilic acyl substitution where the carbonyl compound is used as an electrophile and is attacked by a nucleophilic alcohol. However, other processes are possible; esterification by alkylation reverses the roles of "classic" carbonyl chemistry: a carboxylate anion is used as a nucleophile that displaces a halide ion in an SN2 reaction. With Acid hydrolysis using sulphuric acid and water (equilibrium reaction), the ester splits into a carboxylic acid and alcohol, protons are donated from the acid. The solution can be then distilled and the remaining acid can be checked using UV indicator. Positive results for the test for ester yields a burgundy color. Based on Table 1, the first and second eluate yielded yellow solution which is a negative result for ester while the third eluate gave a burgundy solution which is a positive result and shows the presence of ester. Test for Glycerol (Acrolein Test) Acrolein test is a test for the presence of glycerin or fats. A sample is heated with potassium bisulfate, and acrolein is released if the test is positive. When a fat is heated strongly in the presence of a dehydrating agent such as KHSO4, the glycerol portion of the molecule is dehydrated to form the unsaturated aldehyde, acrolein (CH2=CH-CHO), which has the peculiar odor of burnt grease.

Based on the results that were culled (Table 1), the first second and third eluate did not produce any odor hence indicates the absence of glycerol for each eluates. Test for cholesterol (LiebermanBurchard Test) The Lieberman-Burchard or acetic anhydride test is used for the detection of cholesterol. The formation of a green or greenblue color after a few minutes is positive. Lieberman-Burchard is a reagent used in a colorimetric test to detect cholesterol, which gives a deep green color. This color begins as a purplish, pink color and progresses through to a light green then very dark green color. The color is due to the hydroxyl group (-OH) of cholesterol reacting with the reagents and increasing the conjugation of the unsaturation in the adjacent fused ring. Based on the results that were culled (Table 1), the first and third eluate did not produce any change in color. The second eluate produced a greenish color which indicated the presence of cholesterol.

Table 2: Results of Test for Unsaturation with I2 st nd rd 1 eluate 2 eluate 3 eluate Number of 80 84 6 I2 drops Table 2 shows the actual results for lipid unsaturation with iodine. The test for unsaturation with iodine identifies the level of saturation and the number of bonds an oil, fat or lipid has. The more unsaturated, multi-bonded, the lipid is, the more it absorbs iodine. The table shows that the most unsaturated lipid is the second eluate, and the most saturated lipid is the third eluate. Canola oil is used as a standard for the test for unsaturation.

Canola oil 13

REFERENCES FROM BOOKS:
Barreto , M.C. (2005). Lipid extraction and cholesterol quantification:A simple protocol. Journal of Chemical Education 82(1), 103-104. Bernas ,G.C., Ysrael, M.C., &Bernaldez, A.T. (1994). rd Basic laboratory studies in biochemistry (3 ed.). Manila: UST publishing house. Bettelheim,F.A., March,J. (1990). Introduction to organic and biochemistry. Philadelphia: Saunders College. Campbell, M., Farrell, S. (2012). Biochemistry. 7 ed. China: China Translation & Printing Services Limited. Crisostomo, A., Daya, M., de Guia, R., et.al. (2010). Laboratory Manual in General Biochemistry. Quezon City: C&E Publishing Inc. 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. The Biochemistry Faculty. (1980). A laboratory manual for biochemistr. Quezon City: C.A.S. University of the Philippines
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