Qualitative Analysis of Lipids through the Extraction of Total Lipids from Chicken Egg Yolk

Tarrazona, Yssa Nicole, Tolentino, Gerone Anne, Trinidad, Sofia Blanca, Vallangca, Ma. Jessa Victoria, Villanueva, Kristine Nicole

ABSTRACT
The experiment was done to identify the characteristics of lipids through qualitative analysis. Lipids are amphiphilic substances because of their long, hydrophobic tail and hydrophilic head which consists of a carboxyl group. To attain the objectives of the experiment, total lipids were extracted from chicken egg yolk using ethanol, hexane, and acetone. The extracted polar lipids were then subjected to different qualitative tests, which were test for ester, Acrolein test, Liebermann-Burchard test, and test for lipid unsaturation with Br2. The ethanol extract and the hexane extract rendered a positive result for test for ester and Acrolein test, and Krauts test. For Liebermann-Burchard test, only the acetone extract rendered a positive result. As for the test for lipid unsaturation with Br2, the ethanol extract turned out to be the most unsaturated while the acetone extract is the most saturated. Therefore, it is observed that the first eluate is triacylglycerol, the second eluate is cholesterol, and the third eluate is lecithin.

INTRODUCTION
Lipids are hydrophobic and could be either
fats and oils, waxes, phospholipids, steroids (like cholesterol), and some other related compounds. [1] They are heterogenous and are synthesized by living cells that are soluble in non-polar organic solvents and insoluble in water. [2] Triacylglycerols (fats and oils) serves as a store of energy, which can be released rapidly on demand, and as a reserve of essential fatty acids and precursors for eicosanoids. Many cell types and organs have the ability to synthesise triacylglycerols, but in animals the liver, intestines and adipose tissue are most active for it. [3] On the other hand, cholesterol Cholesterol is a waxy steroid and is transported in the blood plasma of all animals. It is the main sterol synthesized by animals. It builds and maintains cell membranes and is responsible for cell permeability. [4] Lecithin, in contrast, is a fat that is essential in the cells of the body and can be found in many foods, including soybeans and egg yolks. [5] To know the contents of the lipids extracted, it must undergo qualitative tests. To test for the presence of ester, the test for ester was performed; to test for the presence of glycerol, the Acrolein test was performed; and to test for cholesterol, the Liebermann-Burchard test was performed. The experiment must be able to attain the following objectives: (1) to extract total lipids from chicken egg yolk (2) to identify lipids present in each of the fractions using qualitative tests (3)

to determine the degree of unsaturation if lipids through bromine test. [2]

METHODOLOGY
A. Extraction of Total Lipids from Chicken Egg Yolk An equal amount of ethanol was added and mixed to the egg yolk to dehydrate and partially extract the polar lipids. Hexane was then added and mixed until two layers formed. The mixture resulted in fractions of polar and neutral lipids. The upper polar fraction was removed and an equal amount of acetone was added to further precipitate the polar lipids from residual neutral ones, especially cholesterol. The upper layer was then collected and transferred into a clean test tube. B. Test for Ester 10 drops of the eluates were put into different test tubes. 0.5 mL ethanol:1-butanol (3:1) was added to each test tubes, then 2 drops each of 2 M NH2OH HCl and 3 M NaOH was added sequentially and mixed well to the test tubes. The samples were allowed to stand for 5 minutes, then 2 drops of 6 M HCl and 1 drop 5% FeCl36H2O were added and mixed well to 0.1 M HCl. Burgundy coloration will be observed.

C. Test for Glycerol (Acrolein Test) A pinch amount of KHSO4 was added to 10 drops in each of the three test tubes containing the eluates. The test tubes were heated in a boiling water bath. A burnt fat odor indicates the presence of glycerol.

D. Test for Cholesterol Burchard Test)

(Liebermann-

To 10 drops of eluate in each of the 3 test tubes, 0.25 mL dichloromethane was added. After which, 6 drops of acetic anhydride and 2 drops of concentrated H2SO4 were added and mixed well. A greenish color produced after a few minutes would indicate the presence of cholesterol. E. Test for Lipid Unsaturation with Br2 To 10 drops of eluate in each of the 3 test tubes, 3 mL dichloromethane was added and mixed well. Under a fume hood, 5% Br2 in dichloromethane was added dropwise into the test tubes, shaking after each addition until a reddish brown color is achieved. The number of drops of 5% Br2 in dichloromethane was then recorded. The procedure was repeated and the result was compared to 8 drops of coconut oil.

RESULTS AND DISCUSSION

On the experiment performed in identifying the characteristics of the lipids extracted through qualitative analysis, the following results were obtained.
Table 1. Data obtained for the Qualitative Tests Chemical Test for Acrolein LiebermannEster Test Burchard Test 1st Eluate (+) (+) (-) (Ethanol) Burgundy Burnt fat Colorless color odor 2nd Eluate (+) (+) (-) (Hexane) Burgundy Burnt fat Colorless color odor rd 3 Eluate (-) (-) (+) (Acetone) Colorless No burnt Brilliant fat odor green

The test for ester was performed to check whether the eluates contained an ester. The burgundy coloration was due to the carboxylate esters being converted to hydroxamic acids which form a magentacolored complex with ferric ion. Thus, based on the table, it could then be concluded that the 1st and 2nd eluates contain an ester. The Acrolein test was performed to determine which of the eluates contain glycerol. The burnt fat odor is due to the oxidative dehydration of glycerol to acrolein when heated with solid potassium hydrogen sulfate. Thus, based on the table, the 1st and 2nd eluates contain glycerol. The Liebermann-Burchard test was performed to determine which of the eluates contain cholesterol. The brilliant green coloration 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.

Table 2. Data obtained for Test for Lipid Unsaturation with Br2 Eluate Coconut Oil Number 1st 2nd 3rd of Br2 55 40 15 50 drops

This test was performed to determine which of the following eluates was the most saturated. Based on the results obtained, the ethanol extract is the most unsaturated. This was compared to coconut oil which was also shown to be unsaturated. The ethanol extract is the most unsaturated because it absorbed the most number of bromine.

CONCLUSION
Based on the results obtained, it can be concluded that the first eluate is triglyceride,

the second eluate is cholesterol, and the third eluate is licethin, which is a phospholipid.

REFERENCES
1. Lipids: Fats, Oils, Waxes, etc. 03/02/2014
http://biology.clc.uc.edu/courses/bi o104/lipids.htm

2. Crisostomo, A.C. et. al. (2010). Laboratory Manual in General Biochemistry. Quezon City: C&E Publishing, Inc. 3. The AOCS Lipid Library. Triacylglycerols. 03/02/2014 http://lipidlibrary.aocs.org/Lipids /tag2/index.htm 4. MNT Knowledge Center. What is
cholesterol? cholesterol? What causes high

03/02/2014 http://www.medicalnewstoday.c om/articles/9152.php

5. WebMD. Lecithin. 03/02/2014 http://www.webmd.com/vitami nssupplements/ingredientmono966LECITHIN.aspx?activeIngredientI d=966&activeIngredientName=L ECITHIN 6. Mansfield. Qualitative Tests for Lipids. 03/02/2014 http://faculty.mansfield.edu/bga nong/biochemistry/lipids3.htm 7. Oxford Reference. Acrolein test. . 03/02/2014 http://www.oxfordreference.co m/view/10.1093/oi/authority.20 110803095348108