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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
III. three times the caloric weight of the egg white. It contains approximately 60 calories.51g of total fat. . 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.Phospholipids are modified triglycerides because of the one fatty acid chain that is replaced by a phosphate group. E and K) are found in the egg yolk.61g carbohydrates. The yolk of a chicken egg makes up about 33% of the liquid weight of the egg. One large egg (50 grams in weight. It stabilizes animal cell membranes. in vitamin D synthesis and in the synthesis of bile. 17 grams of egg yolk) contains approximately 2. and 4. The yellow color of the yolk is because it contains carotenoids that are known as xanthophylls specifically lutein and zeaxanthin. 0. 210mg cholesterol. (USDA National Nutrient Database) All of the fat soluble vitamins (A. Methodology And cholesterol which has a four interconnected carbon rings.7g protein. Egg yolks are one of the few foods that contain vitamin D naturally. D. The body uses it to synthesize steroid hormones such as sex hormones and hormones of the adrenal cortex. and the water insoluble(hydrophobic) tails face each other. Two layers of phospholipid molecules self-assemble so that their water soluble(hydrophilic) heads form the surface and interior of the membrane.
is added. The mixtures were let stand for 5 minutes. 6-10 drops of ninhydrin solution is added. 2 drops of 2M NH2OH HCl and 3M NaOH is added and mixed well. The upper polar fraction is then collected and with the same amount as the collected upper fraction. after spots appear the plates are placed in hot plate to remove excess I2.1M HCl were added and were mixed well. acetone is added to precipitate the polar lipids from cholesterol and other residual neutral ones. the plates are placed in the container with the I2 vapor.5ml ethanol:1-butanol(3:1) is added to each. Develop the plates first in the solvent mixture of petroleum ether: methanol: water(65:25:4). When the solvent front is ¼ from the top edge. with the same amount. Test for Glycerol (Acrolein Test) 10 drops of the 3 different extracts are placed in separate test tubes and a pinch of KHSO4is added. mixed and let stand for 5 minutes. 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. The upper layer is collected to be used for 2-D Thin Layer Chromatography. Place the three TLC plates on the hot plate for approximately 3minutes to reactivate the silica and then remove them. With the same amount of egg yolk ethanol is first added then mixed.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. 0. Test for Cholesterol (LiebermannBurchard Test) 10 drops of the three different extracts are placed in separate test tubes. Sequentially. put I2 crystals and cover to saturate the container with I2 vapor for 5 minutes. The plates are then totally sprayed with ninhydrin and are placed in the hot plate for 1 minute for the visible result to appear. 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. do the same for spotting of cholesterol and lecithin. After the development on the second solvent mixture. Test for Ester 10 drops of ethanol.25ml of CH2Cl2 is added. 0. 2 drops of 6M HCl and 1 drop 5%FeCl3•6H2O in 0. In a separate beaker. hexane. rotate the plate 90° to the right and transfer to the second solvent mixture of petroleum ether: methanol: NH4OH(65:25:4). H2SO4 is added and is mixed well. Spot the hexane extract on the first TLC plate at least 1cm from the edge of the plate. and acetone extract are placed in separate test tubes. The test tubes are heated in a boiling water bath. 6 drops of acetic anhydride nad 2 drops of conc. When the polar lipids are partially extracted the hexane. Test for Lipid Unsaturation with Br2 .
there are no α-amino acids in all three. TLC of Lecithin Table2. The TLC of lecithin is the only one that showed a development of its sample. 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 α-amino acids are present in the ninhydrin test. 5% Br2 in CH2Cl2 is added dropwise shaking the test tube after each addition. 3ml of CH2Cl2 is added and mixed well. coconut oil and oil sample from the lab. Water bath for acrolein test Figure2. IV. This result indicates that there is an absence of glycerine in the samples. 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. Test for Gycerol (Acrolein Test) Figure4. This means that eluate 3 is positive for the presence of ester. Under the fume hood. 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.10 drops of the 3 different extracts are placed in separate test tubes. 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. TLC of hexane extract Table1. Results and Discussion Thin-Layer Chromatography: Analysis of Lipids from Egg Yolk Figure1. This is also done to 8 drops of canola oil. TLC of Cholesterol Figure3. Therefore. 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 .
Liebermann-Burchard Test Table4. Result for Liebermann-Burchard Test Sample Eluate 1 Eluate 2 Table3. 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. 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. Lipis Unsaturation with Br2 . as it is dehydrated it will form an unsaturated aldehyde which is acrolein. eluate 3 became a yellow solution and eluate 2 produced a blue-green solution which is indicative of a positive result.as KHSO4. The absence of the blue-violet solution indicates the absence of α-amino acids in the samples. Eluate 2 indicates that cholesterol is present in the solution. Result for ninhydrin test Figure6. Test for Lipid Unsaturation with Br2 Figure7. Test for Cholesterol Burchard test) (Liebermann- Eluate 1 did not change in color. These results indicate a negative result to all the samples. This test detects the presence of α-amino acids by oxidative deamination followed by carboxylation. Test for Glycerol (Ninhydrin Test) Figure5.
Chromatography.clc. Freeman. 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.edu/bga nong/biochemistry/pcpurif.com/Objects/ViewObject. A. This test identifies the level of saturation and the number of bonds a lipid has. March. Biochemistry: The Molecular Basis of Life.edu/ faculty/reusch/VirtTxtJml/lipids. Any more errors are due to the extracts or the measuring of reagents put in the specific tests. J. Waxes etc.Boston: McGraw Hill. V.htm retrieved: March 5.(1990). Possible errors in this performed experiment are more likely on the Thin-layer chromatography. Websites: Biomolecules: The Lipids http://www.htm Retrieved: March 5. 2012 Purification of Lipids from Egg Yolk http://faculty.a spx?ID=AP13204 retrieved: March 5.H.L. 2012 Lipids: Fats. E.(2008).wisconline.edu/courses/ bio104/lipids. Philadelphia: Saunders College. McKee. (1967). Introduction to Organic and Biochemistry. http://biology. multi-bonded the lipid is the greater is its absorbance of bromine.h tm Received: March 5.mansfield. Lehninger.Table5. Heftman.chemistry.msu. New York: W. Oils.uc.(2003). New York: Reinhold Publishing Corporation. F. 2012 • • • • • • • . The more unsaturated. 2012 Lipids http://www2.Principles of Biochemistry.A. References • • Books: Bettelheim.
layer Lipids Chromatography of http://lipidlibrary.org/topics/ tlc/index.• Thin. 2012 .aocs.htm Received: March 5.