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THE EXTRACTION AND ANALYSIS OF LIPIDS IN EGG YOLK USING THIN LAYER CHROMATOGRAPHY, COLUMN CHROMATOGRAPHY AND DIFFERENT QUALITATIVE TESTS Nano, Lizette A., Olivete, Lesli Linka Mel L., Ong, Feliz Jem V., Ong, Ralph Timothy S., Ortega, Aira Marie A.* University of Santo Tomas, Faculty of Pharmacy Date submitted: February 18, 2013
Abstract Lipids are molecules that contain hydrocarbons and make up the building blocks of the structure and function of living cells. Lipids are not soluble in water. They are non-polar and are thus soluble in non-polar compounds like chloroform. In the experiment, lipids were extracted from the chicken egg yolk. Lipids are soluble in organic solvents like ethanol. The group used ethanol to partially extract the polar lipids present in the egg yolk. This will result in a two-layer form. The upper layer was then subjected to following different test: TLC (Thin Layer Chromatography) and CC (Column Chromatography. The eluates from the Column Chromatography were then subjected to the following tests: Test for Glycerol, Test for Cholesterol (Liebermann-Burchard Test) and Test for Unsaturation with Bromine. Introduction Lipids are molecules that contain hydrocarbons and make up the building blocks of the structure and function of living cells. Examples of lipids include fats, oils, waxes, certain vitamins, hormones and most of the non-protein membrane of the cells. Lipids are not soluble in water. They are non-polar and are thus soluble in non-polar compounds like chloroform. Since water is a polar environment, it is not soluble in such. Lipids have mainly hydrocarbons in their composition and are highly reduced forms of carbon. When these are metabolized, these are oxidized to release large amounts of energy that are useful to living organisms. Lipids are extracted from plants and animals using non-polar solvents such as ether, chloroform and acetone. There are a number of
classification schemes for lipids. Categorizing them by their functions and structure will give: a. Hydrolyzable/Non-hydrolyzable lipids b. Fatty acids c. Waxes/Fats and oils d. Mono/polyunsaturated and saturated A. Hydrolyzable/Non-hydrolyzable Lipids Lipids that contain a functional group ester are hydrolysable in water. These include neutral fats, waxes, phospholipids, and glycolipids. Nonhydrolyzable lipids lack such functional groups and include steroids and fat-soluble vitamins (e.g. A, D, E, and K). Fats and oils are composed of triacylglycerols or triglycerides. These
8%) ‐ Phosphatidylinositol (0.2. The number of carbon atoms are almost always an even number and are usually unbranched. Waxes/ Fats and oils These are esters with long-chain carboxylic acids and long-alcohols. The objectives of the experiment are as follows: 1. Oleic acid is the most abundant fatty acid in nature. fats are mainly present in animals. To identify lipids present in each of the fractions using qualitative tests 4. Unsaturated fats assume a particular geometry that prevents the molecules from packing as efficiently as they do in saturated molecules. Fatty Acids Fatty acids are long chain carboxylic acids (typically 16 or more carbon atoms) which may or may not contain carbon-carbon double bonds. oils are mainly present in plants and sometimes in fish. C. Mono/Poly. are also found in the egg yolk. Thus the boiling points of unsaturated fats are lower. To determine the degree of unsaturation of lipids through bromine tests. lutein and zeaxanthin. which are fat soluble vitamins. B. Those that have two or more double bonds are called polyunsaturated.2%) ‐ Sphingomyelin (0. Oils are triglycerides that appear as a liquid at room temperature. The following lipids are found: Neutral Lipids (65%) Phospholipids (30%) ‐ PC (25%) ‐ PE (4. To analyse the lipids present in the crude extract using twodimensional thin layer chromatography and column chromatography 3. The lipid content comprises the 31% of the yolk. And then further analysing them through various tests like Thin Layer and Column Chromatography and other qualitative tests using different chemical reagents. E and K. zeaxanthin) -Vitamins A.3trihydroxypropane) and 3 fatty acids to form a triester.are composed of glycerol (1. The egg yolk is comprised of the 30%33% of the total weight of the egg. the group will be extracting the lipids from the egg yolk. To extract total lipids from chicken egg yolk 2. Oleic acid is monounsaturated. Fat is the name given to a class of triglycerides that appear as solid or semisolid at room temperature. The distinctive yellow colored appearance of the egg yolk is caused by a few of its lipid content. In the experiment. D. Procedures . while unsaturated fats are liquids and usually extracted from plants.15%) Cholesterol (5%) Carotenoids (carotenes) Xantophylls (lutein. D. Saturated fats are typically solids and are derived from animals.unsaturated and saturated Those fatty acids with no carbon-carbon double bonds are called saturated.
4. A blue violet coloration indicates the presence of alpha-amino acids in the ninhydrin test. Place the plates on a hot plate to remove excess iodine. Remove the TLC plates when the solvent front is almost ¼ inch from the top and transfer them to the second solvent mixture 6. mix and let it stand for 5 minutes until two layers form. The glass column should have a tapered end plugged with a glass wool. Additional heating will char unsaturated compounds. Prepare a small column by pouring a slurry of 0. Thin Layer Chromatography Analysis of Lipids from Egg Yolk 1. This will result in fractions of polar and neutral lipids. 2. Develop the plate in the first solvent mixture 65:25:4 (v/v/v) petroleum ether:methanol:water. Completely spray the TLC plates with ninhydrin. C. Perform two-dimensional thin layer chromatography and column chromatography on the upper layer B. 9. 65:25:4 (v/v/v) petroleum ether:methanol:water b. Put I2 crystals in a separate beaker and saturate the container with I2 vapor for 5 minutes. 3. Extraction of Total Lipids from Chicken Egg Yolk 1. Spot the extract (from the extraction of total lipids from chicken egg yolk) at least 1cm from the edge of the TLC plates. collecting the eluate in the same test tube as the run-through 4. Remove TLC plates from heat. 65:25:4 (v/v/v) petroleum ether:methanol:NH4OH.5 g silica gel in 4 mL of petroleum ether into a glass column (Pasteur pipette). Completely spray the plate with phosphorus. saving the run-through in a clean test tube. 7. Equilibrate the TLC Solvent mixtures in two separate beakers: a. Place two clean TLC plates on the hot plate with silica side up for approximately 3 minutes to reactivate silica. Phosphorus containing compounds will appear ad blue spots on white background. 65:25:4 (v/v/v) petroleum ether:methanol:NH4OH 2. Add an equal amount of ethanol and mix to dehydrate and partially extract the polar lipids 2. 5. Column Chromatography of Lipids 1. 4. Wash the column with 5 mL 9:1 mixture of petroleum ether :ethyl ether. Add hexane.A. Wash the column with the second eluent (5 mL 5% . Place them on the hot plate for one minute. 8. Pour 1 mL lipid extract (collected from the extraction of total lipids from chicken egg yolk) into the column. 3. Transfer the TLC plates in the beaker with iodine until spots appear. Remove the upper polar fraction and add an equal amount if acetone to further precipitate the polar lipids from residual neutral ones like cholesterol. 3. Place them on the hot plate for 30 seconds. Collect the upper layer and transfer into a clean test tube.
Save the different eluates for qualitative analysis. HCl and 3 M NaOH. Add 3 mL DCM and mix well.5 mL ethanol:1butanol (3:1). Add a pinch amount of KHSO4 to 10 drops of the eluate in a test tube. 3. 2. 5 mL DCM:Methanol:Water (1:3:1) and collect the eluate in another test tube. Test for Glycerol ( Acrolein Test) 1. Add 6 drops acetic anhydride and 2 drops conc. wash the column with the last eluent. Add 0. Heat the test tube in a boiling wather bath and note the odor produced. Warm the test tube and observe if there are any changes. 4. Add 2 drops 6 M HCl and 1 drop 5% FeCl36H2O in 0. Allow the samples to stand for 5 minutes. Under a fume hood. Results and Discussions .1 mL HCl. Place 10 drops eluate in a test tube. Place 10 drops eluate in a test tube. collecting the eluate in another clean test tube. add 5% Br2 in DCM V. Test for Ester 1. corn and olive oil. and mix well. IV. II. and mix well. 3. H2SO4 and mix well.25 mL dichloromethane. A greenish color produced after a few minutes indicates the presence of cholesterol. D. III. 2. Place ten drops of the eluates in separate test tubes. dropwise into the test tub. Qualitative Tests for Lipids I. Add 3 mL of Kraut’s reagent to 10 drops of the eluate in a test tube.methanol in DCM). 2. 3. 5. Test for Cholesterol (Liebermann-Burchard Test) 1. Burnt fat odor indicates the presence of glycerol. Test for Glycerol (Kraut’s Test) 1. Record the added number of drops of 5% Br2 in DCM. Test for Lipid Unsaturation with Br2 1. Repeat the procedure and compare the result with the following: 8 drops each of coconut. shaking after each addition until a reddish brown color persists. canola. 2. Samples with esters will produce a burgundy color. Finally. Add sequentially 2 drops each 2M NH2OH. 6. 5. 2. Add 0. 4.
2-0. the Extraction of Lipids. As one can see. The stationary phases are usually finely ground powders or gels and/or microporous for an increased surface. one can imply that the sample from the upper layer is indeed the polar lipid content of the egg yolk. The mobile phase or eluent is either a pure solvent or a mixture of different solvents. The group plotted a sample from the upper layer formed from the previous experiment. to further separate the polar and neutral lipids. carotenoids. The most common stationary phase for a column chromatography is silica gel. it washed down the most nonpolar component of . This eluent was believed to be a nonpolar solvent. (The component of the lower layer predominantly is cholesterol. The eluent has also been chosen so that the different compounds can be separated effectively. The group added hexane. the sample from the upper layer travelled far than the sample taken from the lower layer. lipids were extracted from the chicken egg yolk.0 TLC Plate (This is just a representation of the actual result) The TLC Solvent mixtures used in the experiment were 65:25:4 (v/v/v) petroleum:ether:methanol:water and 65:25:4 (v/v/v) petroleum:ether: methanol: NH4OH. The first eluent the group used was a 5 mL 9:1 mixture of petroleum ether:ethyl ether. This will result in a two-layer form. From the results. xanthophyll and lecithin. With it. the upper layer is the polar one and will precipitate. Thin Layer Chromatography Separation of polar lipids from the neutral ones is further characterized through adsorption chromatography using mixtures of organic solvents in varying proportions. Lipids are soluble in organic solvents like ethanol. Neutral lipids like cholesterol and triacylglycerols were believed to have been found in the residual neutral lipids.In the experiment. The solvent mixtures served as the mobile phase of the medium and are rendered polar.3 in order to minimize the time and amount of eluent to run the chromatography. The upper layer was then subjected to following different test: TLC (Thin Layer Chromatography) and CC (Column Chromatography. It is chosen so that the retention factor value of the compound of interest is roughly around 0. The eluent is optimized in small scale pretests. Figure 2. and from the residual lower layer. The group used ethanol to partially extract the polar lipids present in the egg yolk. often using thin layer chromatography (TLC) with the same stationary phase. The polar lipids in the material were said to be phospholipids. Column Chromatography The stationary phase of a column chromatography is a solid.) The results are as follows.
The third eluent used was 5 mL DCM:methanol:water (1:3:1). the triacylglycerol. The burnt fat odor indicates the presence of glycerol. A deep green color would indicate a positive result. is a nonpolar solvent. Eluate 1st 2nd 3rd Coconut Oil Canola Oil Number of Br2 drops 55 67 64 24 18 . Kraut’s Test The group was unable to perform this test due to the unavailability of Kaut’s reagent. Tests for Lipids Figure 1. In the group’s experiment. it was eluted 5 the 5% methanol in DCM. The second eluent used was 5mL 5% methanol in DCM. Saponifiable lipids are capable of alkaline hydrolysis of esters of fatty acids to form glycerol and sodium salt of fatty acids commonly known as soap. The third eluate is the only one that yielded a positive result. Test for Lipid Unsaturation with Br2 Figure 3. Suspected errors e. This eluent is a polar solvent and therefore expected to wash down the polar lipids in egg yolk like: lecithin.0 Actual Results of the different tests for Lipids st nd rd Chemical 1 Eluate 2 Eluate 3 Eluate Test Ester Yellow Yellow Burgundy solution solution Solution Glycerol No odor No odor Burnt Fat (Acrolein’s) odor Glycerol (Kraut’s) Cholesterol No color Green negative change Bromine (to be (to be (to be followed) followed) followed) Acrolein Test This test differentiates cholesterol and lecithin. When lipids containing glycerol are heated in the presence of potassium hydrogen sulphate. According to the group’s found references. the next component to be washed down is Cholesterol. This eluent. Due to its nonpolar characteristic. forming acrolein. phospholipids and xantophylls. it is the second eluate (cholesterol-containing) that should have the positive result. Having washed down triacylglycerol. The first eluate was the triacylglycerol. Liebermann-Burchard Test This test is used for the detection of cholesterol. it is the second eluate (cholesterol-containing) that gave the positive results. This is due to the hydroxyl group of cholesterol reacting with the reagents (Acetic anhydride and concentrated sulphuric acid) and increasing the conjugation of the unsaturation in the adjacent fused ring.0 Test for Unsaturation of Lipid Test for Ester This test determines the presence of an ester functional group in a solution. The said polar lipids were believed to be the component of the third eluate. In the experiment. which has an unpleasant odor. too. This test is performed to analyse whether the lipid is saponifiable or non-saponifiable.the extracted lipid (crude lipid). the glycerol is dehydrated.g contamination may have contributed to the erroneous result of this particular experiment. A burgundy colored solution indicates a positive result. the third eluate produced the burnt fat odor.
The number of drops determines the level of saturation.com/doc/51367254/Ac rolein-Test-and-Ester-Test-for-Lipids .aspx http://www.net/health/What-are-Lipids. the more unsaturated it is.pdf http://myweb. resulting to the distinctive red color of the solution.edu/~duahn/te aching/Neobiomaterials%20and%20Bior egulation/Egg%20Components. The fewer drops of the bromine to achieve the red color of the solution. References: http://www.liu.iastate.The bromine test is a qualitative test for the presence of unsaturated carboncarbon double bonds and phenols.edu/~nmatsuna/che4x/e 8lipids. The bromine attaches itself to the double bonds in the unsaturated fatty acid chain.newsmedical.pdf http://www.public.scribd.
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