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Biochemistry Laboratory - Copy

Biochemistry Laboratory - Copy

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Published by: AIra Ortega on Feb 21, 2013
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Biochemistry Laboratory –PH-BIOCHEM (2012 – 2013) 2B-Ph Group 7 Experiment 6

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

E and K. D. Oleic acid is the most abundant fatty acid in nature. which are fat soluble vitamins. Thus the boiling points of unsaturated fats are lower. Those that have two or more double bonds are called polyunsaturated.3trihydroxypropane) and 3 fatty acids to form a triester. The distinctive yellow colored appearance of the egg yolk is caused by a few of its lipid content. The egg yolk is comprised of the 30%33% of the total weight of the egg. And then further analysing them through various tests like Thin Layer and Column Chromatography and other qualitative tests using different chemical reagents. To extract total lipids from chicken egg yolk 2. To determine the degree of unsaturation of lipids through bromine tests. To identify lipids present in each of the fractions using qualitative tests 4. D. B. fats are mainly present in animals. the group will be extracting the lipids from the egg yolk. Oleic acid is monounsaturated. In the experiment.2. Waxes/ Fats and oils These are esters with long-chain carboxylic acids and long-alcohols. oils are mainly present in plants and sometimes in fish. The lipid content comprises the 31% of the yolk. while unsaturated fats are liquids and usually extracted from plants. Unsaturated fats assume a particular geometry that prevents the molecules from packing as efficiently as they do in saturated molecules. Mono/Poly. Fat is the name given to a class of triglycerides that appear as solid or semisolid at room temperature. 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. are also found in the egg yolk.are composed of glycerol (1. The number of carbon atoms are almost always an even number and are usually unbranched. zeaxanthin) -Vitamins A.2%) ‐ Sphingomyelin (0.unsaturated and saturated Those fatty acids with no carbon-carbon double bonds are called saturated. lutein and zeaxanthin.15%) Cholesterol (5%) Carotenoids (carotenes) Xantophylls (lutein. The objectives of the experiment are as follows: 1. Procedures . C.8%) ‐ Phosphatidylinositol (0. Saturated fats are typically solids and are derived from animals. To analyse the lipids present in the crude extract using twodimensional thin layer chromatography and column chromatography 3. 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.

Spot the extract (from the extraction of total lipids from chicken egg yolk) at least 1cm from the edge of the TLC plates. Collect the upper layer and transfer into a clean test tube. Perform two-dimensional thin layer chromatography and column chromatography on the upper layer B. Equilibrate the TLC Solvent mixtures in two separate beakers: a. 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. Remove TLC plates from heat. saving the run-through in a clean test tube. The glass column should have a tapered end plugged with a glass wool. Pour 1 mL lipid extract (collected from the extraction of total lipids from chicken egg yolk) into the column. This will result in fractions of polar and neutral lipids. Thin Layer Chromatography Analysis of Lipids from Egg Yolk 1. Add an equal amount of ethanol and mix to dehydrate and partially extract the polar lipids 2. Put I2 crystals in a separate beaker and saturate the container with I2 vapor for 5 minutes. 4. 2. Place them on the hot plate for one minute. Prepare a small column by pouring a slurry of 0. Transfer the TLC plates in the beaker with iodine until spots appear. Wash the column with the second eluent (5 mL 5% . 3. Place two clean TLC plates on the hot plate with silica side up for approximately 3 minutes to reactivate silica. Place them on the hot plate for 30 seconds. 5. 65:25:4 (v/v/v) petroleum ether:methanol:NH4OH. 4. Phosphorus containing compounds will appear ad blue spots on white background. mix and let it stand for 5 minutes until two layers form. 9. 7. 8. C. Column Chromatography of Lipids 1. Extraction of Total Lipids from Chicken Egg Yolk 1. 3.A. A blue violet coloration indicates the presence of alpha-amino acids in the ninhydrin test. 65:25:4 (v/v/v) petroleum ether:methanol:water b. Add hexane. Develop the plate in the first solvent mixture 65:25:4 (v/v/v) petroleum ether:methanol:water. Remove the upper polar fraction and add an equal amount if acetone to further precipitate the polar lipids from residual neutral ones like cholesterol. Completely spray the plate with phosphorus. 3. 65:25:4 (v/v/v) petroleum ether:methanol:NH4OH 2. Wash the column with 5 mL 9:1 mixture of petroleum ether :ethyl ether. collecting the eluate in the same test tube as the run-through 4.5 g silica gel in 4 mL of petroleum ether into a glass column (Pasteur pipette). Completely spray the TLC plates with ninhydrin. Additional heating will char unsaturated compounds.

4. Results and Discussions . 2. IV. Test for Glycerol ( Acrolein Test) 1. Repeat the procedure and compare the result with the following: 8 drops each of coconut. 3. III. dropwise into the test tub. collecting the eluate in another clean test tube. Test for Glycerol (Kraut’s Test) 1. 5.methanol in DCM). HCl and 3 M NaOH. Under a fume hood. 3. A greenish color produced after a few minutes indicates the presence of cholesterol. 2. Add 2 drops 6 M HCl and 1 drop 5% FeCl36H2O in 0. Test for Lipid Unsaturation with Br2 1. Test for Cholesterol (Liebermann-Burchard Test) 1. wash the column with the last eluent.5 mL ethanol:1butanol (3:1). H2SO4 and mix well. 2. 5.25 mL dichloromethane. Add 0. Allow the samples to stand for 5 minutes. D. 4. Add 3 mL DCM and mix well. 2. Add sequentially 2 drops each 2M NH2OH. Heat the test tube in a boiling wather bath and note the odor produced. Add a pinch amount of KHSO4 to 10 drops of the eluate in a test tube. Test for Ester 1. shaking after each addition until a reddish brown color persists. corn and olive oil. Finally.1 mL HCl. canola. Save the different eluates for qualitative analysis. 6. and mix well. Record the added number of drops of 5% Br2 in DCM. Place ten drops of the eluates in separate test tubes. Samples with esters will produce a burgundy color. Place 10 drops eluate in a test tube. Burnt fat odor indicates the presence of glycerol. 3. add 5% Br2 in DCM V. 5 mL DCM:Methanol:Water (1:3:1) and collect the eluate in another test tube. Add 6 drops acetic anhydride and 2 drops conc. Place 10 drops eluate in a test tube. Warm the test tube and observe if there are any changes. Add 3 mL of Kraut’s reagent to 10 drops of the eluate in a test tube. 2. and mix well. Qualitative Tests for Lipids I. II. Add 0.

As one can see. the Extraction of Lipids. Lipids are soluble in organic solvents like ethanol. the upper layer is the polar one and will precipitate. It is chosen so that the retention factor value of the compound of interest is roughly around 0.0 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 eluent is optimized in small scale pretests. Neutral lipids like cholesterol and triacylglycerols were believed to have been found in the residual neutral lipids. Figure 2.3 in order to minimize the time and amount of eluent to run the chromatography. The mobile phase or eluent is either a pure solvent or a mixture of different solvents. the sample from the upper layer travelled far than the sample taken from the lower layer. it washed down the most nonpolar component of . The polar lipids in the material were said to be phospholipids. The upper layer was then subjected to following different test: TLC (Thin Layer Chromatography) and CC (Column Chromatography.In the experiment.) The results are as follows. lipids were extracted from the chicken egg yolk. With it. From the results. carotenoids. The stationary phases are usually finely ground powders or gels and/or microporous for an increased surface. The most common stationary phase for a column chromatography is silica gel. The group used ethanol to partially extract the polar lipids present in the egg yolk. and from the residual lower layer. one can imply that the sample from the upper layer is indeed the polar lipid content of the egg yolk. The first eluent the group used was a 5 mL 9:1 mixture of petroleum ether:ethyl ether. xanthophyll and lecithin. The eluent has also been chosen so that the different compounds can be separated effectively. The group plotted a sample from the upper layer formed from the previous experiment. The group added hexane. Column Chromatography The stationary phase of a column chromatography is a solid. This will result in a two-layer form. to further separate the polar and neutral lipids.2-0. often using thin layer chromatography (TLC) with the same stationary phase. The solvent mixtures served as the mobile phase of the medium and are rendered polar. 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. (The component of the lower layer predominantly is cholesterol. This eluent was believed to be a nonpolar solvent.

it is the second eluate (cholesterol-containing) that should have the positive result. Eluate 1st 2nd 3rd Coconut Oil Canola Oil Number of Br2 drops 55 67 64 24 18 . This test is performed to analyse whether the lipid is saponifiable or non-saponifiable. A burgundy colored solution indicates a positive result. This eluent is a polar solvent and therefore expected to wash down the polar lipids in egg yolk like: lecithin. which has an unpleasant odor. Test for Lipid Unsaturation with Br2 Figure 3. forming acrolein. it was eluted 5 the 5% methanol in DCM. The burnt fat odor indicates the presence of glycerol. Having washed down triacylglycerol. the triacylglycerol. When lipids containing glycerol are heated in the presence of potassium hydrogen sulphate. The third eluent used was 5 mL DCM:methanol:water (1:3:1). Tests for Lipids Figure 1. According to the group’s found references. The third eluate is the only one that yielded a positive result. the next component to be washed down is Cholesterol.the extracted lipid (crude lipid). the glycerol is dehydrated.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. Suspected errors e. the third eluate produced the burnt fat odor. phospholipids and xantophylls. 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. is a nonpolar solvent. Due to its nonpolar characteristic. A deep green color would indicate a positive result. This eluent. The said polar lipids were believed to be the component of the third eluate. The first eluate was the triacylglycerol. In the group’s experiment. too. The second eluent used was 5mL 5% methanol in DCM. 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. it is the second eluate (cholesterol-containing) that gave the positive results. Kraut’s Test The group was unable to perform this test due to the unavailability of Kaut’s reagent.g contamination may have contributed to the erroneous result of this particular experiment. In the experiment. Liebermann-Burchard Test This test is used for the detection of cholesterol.0 Test for Unsaturation of Lipid Test for Ester This test determines the presence of an ester functional group in a solution.

liu. The number of drops determines the level of saturation.aspx http://www.edu/~nmatsuna/che4x/e 8lipids.scribd.The bromine test is a qualitative test for the presence of unsaturated carboncarbon double bonds and phenols. the more unsaturated it is. resulting to the distinctive red color of the solution.newsmedical. References: http://www.pdf http://www.net/health/What-are-Lipids.com/doc/51367254/Ac rolein-Test-and-Ester-Test-for-Lipids .iastate.pdf http://myweb. The fewer drops of the bromine to achieve the red color of the solution.edu/~duahn/te aching/Neobiomaterials%20and%20Bior egulation/Egg%20Components. The bromine attaches itself to the double bonds in the unsaturated fatty acid chain.public.

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