University of Santo Tomas FACULTY OF PHARMACY Espaa, Manila Analysis of Lipids in Egg Yolk Group III Chiu, Carlo

Ching, Danica Choi, Adrian Raphael Chua, Victor Correa, Pamela Cjisel _________________________________________________________________________ I. Abstract
The experiment is performed by extracting lipids from chicken egg yolk using hexane, ethanol and acetone. Ethanol is added to the egg yolk to dehydrate and partially extract the polar lipids, hexane is added and let stand for 5 minutes. The upper polar fraction is removed and then acetone is added to further precipitate the polar lipids, more importantly the cholesterol in it. In between these extractions, a portion of each extraction is collected to be used for the qualitative tests for lipids such as: Test for Ester, Acrolein Test, LiebermannBurchard Test and Test for Lipid Unsaturation with Br2. The upper layer of the extraction is collected and will be used for the two-dimensional thin-layer chromatography analysis for the lipids in egg yolk.

____________________________________________________________________________ II. Introduction fats, phospholipids and cholesterol. Neutral fats also known as triglycerides are three fatty acid chains attached to a single glycerol molecule by dehydration synthesis. This stores energy fuel, insulates body tissues and cushions and protects organs.

Lipids are a large and diverse group of naturally occurring organic compounds that are related by their solubility in nonpolar organic solvents such as acetone, which is used in extraction of cholesterol in this experiment, and their insolubility in water. All lipids are hydrophobic, these are the fats and oils, waxes, phospholipids, steroids like cholesterol and many others. Three examples of lipids we encounter in this experiment are the neutral

Phospholipids are modified triglycerides because of the one fatty acid chain that is replaced by a phosphate group. Two layers of phospholipid molecules self-assemble so that their water soluble(hydrophilic) heads form the surface and interior of the membrane, and the water insoluble(hydrophobic) tails face each other.

The yolk of a chicken egg makes up about 33% of the liquid weight of the egg. It contains approximately 60 calories, three times the caloric weight of the egg white. One large egg (50 grams in weight, 17 grams of egg yolk) contains approximately 2.7g protein, 210mg cholesterol, 0.61g carbohydrates, and 4.51g of 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 that contain vitamin D naturally. The composition (by weight) of the most prevalent fatty acids found 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: 41% Stearic acid: 4% Myristic acid: 1% Egg yolk is also a source of lecithin an emulsifier and sulfactant. The yellow color of the yolk is because it contains carotenoids that are known as xanthophylls specifically lutein and zeaxanthin. III. Methodology

And cholesterol which has a four interconnected carbon rings. It stabilizes animal cell membranes. The body uses it to synthesize steroid hormones such as sex hormones and hormones of the adrenal cortex, in vitamin D synthesis and in the synthesis of bile.

Reagents and Materials: Silica gel Acetone Hexane 3M NaOH Concentrated H2SO4 6M HCl KHSO4 2M NH2OH HCl 5% FeCl3 Acetic Anhydride Iodine crystals TLC solvent mixtures: Petroleum ether: methanol: water (65:25:4) Petroleum ether: methanol: NH4OH (65:25:4) Ninhydrin spray Extraction of Total Lipids from Chicken Egg Yolk The chicken egg yolk is isolated from the egg whites. With the same amount of egg yolk ethanol is first added then mixed. When the polar lipids are partially extracted the hexane, with the same amount, is added, mixed and let stand for 5 minutes. The upper polar fraction is then collected and with the same amount as the collected upper fraction, acetone is added to precipitate the polar lipids from cholesterol and other residual neutral ones. The upper layer is collected to be used for 2-D Thin Layer Chromatography. Thin Layer Chromatography: Analysis of Lipids from Egg yolk The two solvent mixtures (Petroleum ether: methanol: water (65:25:4) and Petroleum ether: methanol: NH4OH (65:25:4)) are equilibrated into two separate beakers. Place the three TLC plates on the hot plate for approximately 3minutes to reactivate the silica and then remove them. Spot the hexane extract on the first TLC plate at least 1cm from the edge of the plate, do the same for spotting of cholesterol and lecithin. Develop the plates

first in the solvent mixture of petroleum ether: methanol: water(65:25:4). When the solvent front is from the top edge, rotate the plate 90 to the right and transfer to the second solvent mixture of petroleum ether: methanol: NH4OH(65:25:4). In a separate beaker, put I2 crystals and cover to saturate the container with I2 vapor for 5 minutes. After the development on the second solvent mixture, the plates are placed in the container with the I2 vapor, after spots appear the plates are placed in hot plate to remove excess I2. The plates are then totally sprayed with ninhydrin and are placed in the hot plate for 1 minute for the visible result to appear. Test for Ester 10 drops of ethanol, hexane, and acetone extract are placed in separate test tubes. 0.5ml ethanol:1-butanol(3:1) is added to each. Sequentially, 2 drops of 2M NH2OH HCl and 3M NaOH is added and mixed well. The mixtures were let stand for 5 minutes. 2 drops of 6M HCl and 1 drop 5%FeCl36H2O in 0.1M HCl were added and were mixed well. Test for Glycerol (Acrolein Test) 10 drops of the 3 different extracts are placed in separate test tubes and a pinch of KHSO4is added. The test tube is then heated in a boiling water bath. Test for Glycerol (Ninhydrin test) In three separate test tube that contains the extracts, 6-10 drops of ninhydrin solution is added. The test tubes are heated in a boiling water bath. Test for Cholesterol (LiebermannBurchard Test) 10 drops of the three different extracts are placed in separate test tubes. 0.25ml of CH2Cl2 is added. 6 drops of acetic anhydride nad 2 drops of conc. H2SO4 is added and is mixed well. Test for Lipid Unsaturation with Br2

10 drops of the 3 different extracts are placed in separate test tubes. 3ml of CH2Cl2 is added and mixed well. Under the fume hood, 5% Br2 in CH2Cl2 is added dropwise shaking the test tube after each addition. This is also done to 8 drops of canola oil, coconut oil and oil sample from the lab. IV. Results and Discussion Thin-Layer Chromatography: Analysis of Lipids from Egg Yolk
Figure1. TLC of hexane extract

Table1. result for test for ester

Sample Eluate 1 Eluate 2 Eluate 3

Result Yellow solution Yellow solution Burgundy solution

Eluate 3 yielded the positive result which is the formation of the burgundy solution while 1 and 2 yielded a yellow solution which is a negative result. This means that eluate 3 is positive for the presence of ester. An alcohol and a carboxylic acid undergoes esterification to yield an ester and acid hydrolysis to split the ester to an alchol and carboxylic acid. Test for Gycerol (Acrolein Test)
Figure4. Water bath for acrolein test

Figure2. TLC of Cholesterol

Figure3. TLC of Lecithin

Table2. result of acrolein test

Sample Eluate 1 Eluate 2 A blue-violet coloration is absent in all three of the thin-layer chromatography that should indicate whether the -amino acids are present in the ninhydrin test. Therefore, there are no -amino acids in all three. The TLC of lecithin is the only one that showed a development of its sample. Qualitative Tests for Lipids Test for Ester Eluate 3

Result No odor No odor No odor

All the samples yielded no odor therefore having a negative result for the acrolein test. This result indicates that there is an absence of glycerine in the samples. Burnt fat odor is observed for a positive result due to the fat that is strongly heated in the presence of a dehydrating agent such

as KHSO4, as it is dehydrated it will form an unsaturated aldehyde which is acrolein. Test for Glycerol (Ninhydrin Test)
Figure5. Result for ninhydrin test

Figure6. Liebermann-Burchard Test

Table4. Result for Liebermann-Burchard Test

Sample Eluate 1 Eluate 2

Table3. result of ninhydrin test

Result No color Blue-green soln Yellow soln

Eluate 3 Result orange

Sample Eluate 1 Eluate 2 Eluate 3

Redsolution No color No color

Eluate 1 produced a red-orange solution while 2 and 3 did not produce any color. These results indicate a negative result to all the samples. The absence of the blue-violet solution indicates the absence of -amino acids in the samples. This test detects the presence of -amino acids by oxidative deamination followed by carboxylation. Test for Cholesterol Burchard test) (Liebermann-

Eluate 1 did not change in color, eluate 3 became a yellow solution and eluate 2 produced a blue-green solution which is indicative of a positive result. Eluate 2 indicates that cholesterol is present in the solution. This color is because of the hydroxyl group of cholesterol reacting with the reagents and increasing the conjugation of the unsaturation o the adjacent fused ring. Test for Lipid Unsaturation with Br2
Figure7. Lipis Unsaturation with Br2

Table5. Drops of Br2 for lipid unsaturation

Sample Eluate 1 Eluate 2 Eluate 3 Canola oil Coconut oil Oil sample

Drops of Br2 15 15 15 55 20 10

The most unsaturated among the samples is the canola oil. This test identifies the level of saturation and the number of bonds a lipid has. The more unsaturated, multi-bonded the lipid is the greater is its absorbance of bromine. Possible errors in this performed experiment are more likely on the Thin-layer chromatography. Any more errors are due to the extracts or the measuring of reagents put in the specific tests. V. References Books: 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).Principles of Biochemistry. New York: W.H. Freeman. McKee.(2003). Biochemistry: The Molecular Basis of Life.Boston: McGraw Hill. Websites: Biomolecules: The Lipids http://www.wisconline.com/Objects/ViewObject.a spx?ID=AP13204 retrieved: March 5, 2012 Purification of Lipids from Egg Yolk http://faculty.mansfield.edu/bga nong/biochemistry/pcpurif.htm retrieved: March 5, 2012 Lipids: Fats, Oils, Waxes etc. http://biology.clc.uc.edu/courses/ bio104/lipids.htm Retrieved: March 5, 2012 Lipids http://www2.chemistry.msu.edu/ faculty/reusch/VirtTxtJml/lipids.h tm Received: March 5, 2012

Thin- layer Lipids

Chromatography

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http://lipidlibrary.aocs.org/topics/ tlc/index.htm Received: March 5, 2012