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

are composed of glycerol (1,2,3trihydroxypropane) and 3 fatty acids to form a triester. B. 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. The number of carbon atoms are almost always an even number and are usually unbranched. Oleic acid is the most abundant fatty acid in nature. C. Waxes/ Fats and oils These are esters with long-chain carboxylic acids and long-alcohols. Fat is the name given to a class of triglycerides that appear as solid or semisolid at room temperature, fats are mainly present in animals. Oils are triglycerides that appear as a liquid at room temperature, oils are mainly present in plants and sometimes in fish. D. Mono/Poly- unsaturated and saturated Those fatty acids with no carbon-carbon double bonds are called saturated. Those that have two or more double bonds are called polyunsaturated. Oleic acid is monounsaturated. Saturated fats are typically solids and are derived from animals, 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. Thus the boiling points of unsaturated fats are lower. The egg yolk is comprised of the 30%33% of the total weight of the egg. The lipid content comprises the 31% of the yolk. The following lipids are found:

Neutral Lipids (65%) Phospholipids (30%) PC (25%) PE (4.2%) Sphingomyelin (0.8%) Phosphatidylinositol (0.15%) Cholesterol (5%) Carotenoids (carotenes) Xantophylls (lutein, zeaxanthin) -Vitamins A, D, E and K, which are fat soluble vitamins, are also found in the egg yolk. The distinctive yellow colored appearance of the egg yolk is caused by a few of its lipid content, lutein and zeaxanthin. In the experiment, the group will be extracting the lipids from the egg yolk. And then further analysing them through various tests like Thin Layer and Column Chromatography and other qualitative tests using different chemical reagents. The objectives of the experiment are as follows: 1. To extract total lipids from chicken egg yolk 2. To analyse the lipids present in the crude extract using twodimensional thin layer chromatography and column chromatography 3. To identify lipids present in each of the fractions using qualitative tests 4. To determine the degree of unsaturation of lipids through bromine tests. Procedures

A. Extraction of Total Lipids from Chicken Egg Yolk 1. Add an equal amount of ethanol and mix to dehydrate and partially extract the polar lipids 2. Add hexane, mix and let it stand for 5 minutes until two layers form. This will result in fractions of polar and neutral lipids. 3. Remove the upper polar fraction and add an equal amount if acetone to further precipitate the polar lipids from residual neutral ones like cholesterol. 4. 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. Thin Layer Chromatography Analysis of Lipids from Egg Yolk 1. Equilibrate the TLC Solvent mixtures in two separate beakers: a. 65:25:4 (v/v/v) petroleum ether:methanol:water b. 65:25:4 (v/v/v) petroleum ether:methanol:NH4OH 2. Place two clean TLC plates on the hot plate with silica side up for approximately 3 minutes to reactivate silica. Remove TLC plates from heat. 3. Spot the extract (from the extraction of total lipids from chicken egg yolk) at least 1cm from the edge of the TLC plates. 4. Develop the plate in the first solvent mixture 65:25:4 (v/v/v) petroleum ether:methanol:water. 5. Remove the TLC plates when the solvent front is almost inch from the top and transfer them to the second solvent mixture

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65:25:4 (v/v/v) petroleum ether:methanol:NH4OH. Put I2 crystals in a separate beaker and saturate the container with I2 vapor for 5 minutes. Transfer the TLC plates in the beaker with iodine until spots appear. Place the plates on a hot plate to remove excess iodine. Completely spray the TLC plates with ninhydrin. Place them on the hot plate for one minute. A blue violet coloration indicates the presence of alpha-amino acids in the ninhydrin test. Completely spray the plate with phosphorus. Place them on the hot plate for 30 seconds. Phosphorus containing compounds will appear ad blue spots on white background. Additional heating will char unsaturated compounds.

C. Column Chromatography of Lipids 1. Prepare a small column by pouring a slurry of 0.5 g silica gel in 4 mL of petroleum ether into a glass column (Pasteur pipette). The glass column should have a tapered end plugged with a glass wool. 2. Pour 1 mL lipid extract (collected from the extraction of total lipids from chicken egg yolk) into the column, saving the run-through in a clean test tube. 3. 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. Wash the column with the second eluent (5 mL 5%

methanol in DCM), collecting the eluate in another clean test tube. 5. Finally, wash the column with the last eluent, 5 mL DCM:Methanol:Water (1:3:1) and collect the eluate in another test tube. 6. Save the different eluates for qualitative analysis. D. Qualitative Tests for Lipids I. Test for Ester 1. Place ten drops of the eluates in separate test tubes. 2. Add 0.5 mL ethanol:1butanol (3:1). 3. Add sequentially 2 drops each 2M NH2OH, HCl and 3 M NaOH, and mix well. 4. Allow the samples to stand for 5 minutes. 5. Add 2 drops 6 M HCl and 1 drop 5% FeCl36H2O in 0.1 mL HCl, and mix well. Samples with esters will produce a burgundy color. II. Test for Glycerol ( Acrolein Test) 1. Add a pinch amount of KHSO4 to 10 drops of the eluate in a test tube. 2. Heat the test tube in a boiling wather bath and note the odor produced. Burnt fat odor indicates the presence of glycerol. Test for Glycerol (Krauts Test)

1. Add 3 mL of Krauts reagent to 10 drops of the eluate in a test tube. 2. Warm the test tube and observe if there are any changes. IV. Test for Cholesterol (Liebermann-Burchard Test) 1. Place 10 drops eluate in a test tube. 2. Add 0.25 mL dichloromethane. 3. Add 6 drops acetic anhydride and 2 drops conc. H2SO4 and mix well. A greenish color produced after a few minutes indicates the presence of cholesterol. Test for Lipid Unsaturation with Br2
1. Place 10 drops eluate in a test tube. 2. Add 3 mL DCM and mix well. 3. Under a fume hood, add 5% Br2 in DCM

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dropwise into the test tub, shaking after each addition until a reddish brown color persists. Record the added number of drops of 5% Br2 in DCM. 4. Repeat the procedure and compare the result with the following: 8 drops each of coconut, canola, corn and olive oil. Results and Discussions

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. The polar lipids in the material were said to be phospholipids, carotenoids, xanthophyll and lecithin. The group added hexane, to further separate the polar and neutral lipids. This will result in a two-layer form; the upper layer is the polar one and will precipitate. Neutral lipids like cholesterol and triacylglycerols were believed to have been found in the residual neutral lipids. The upper layer was then subjected to following different test: TLC (Thin Layer Chromatography) and CC (Column Chromatography. 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 group plotted a sample from the upper layer formed from the previous experiment, the Extraction of Lipids, and from the residual lower layer. (The component of the lower layer predominantly is cholesterol.) The results are as follows.
Figure 2.0 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 solvent mixtures served as the mobile phase of the medium and are rendered polar. As one can see, the sample from the upper layer travelled far than the sample taken from the lower layer. From the results, one can imply that the sample from the upper layer is indeed the polar lipid content of the egg yolk. Column Chromatography The stationary phase of a column chromatography is a solid. The most common stationary phase for a column chromatography is silica gel. The stationary phases are usually finely ground powders or gels and/or microporous for an increased surface. The mobile phase or eluent is either a pure solvent or a mixture of different solvents. It is chosen so that the retention factor value of the compound of interest is roughly around 0.2-0.3 in order to minimize the time and amount of eluent to run the chromatography. The eluent has also been chosen so that the different compounds can be separated effectively. The eluent is optimized in small scale pretests, often using thin layer chromatography (TLC) with the same stationary phase. The first eluent the group used was a 5 mL 9:1 mixture of petroleum ether:ethyl ether. This eluent was believed to be a nonpolar solvent. With it, it washed down the most nonpolar component of

the extracted lipid (crude lipid), the triacylglycerol. The first eluate was the triacylglycerol. The second eluent used was 5mL 5% methanol in DCM. This eluent, too, is a nonpolar solvent. Having washed down triacylglycerol, the next component to be washed down is Cholesterol. Due to its nonpolar characteristic, it was eluted 5 the 5% methanol in DCM. The third eluent used was 5 mL DCM:methanol:water (1:3:1). This eluent is a polar solvent and therefore expected to wash down the polar lipids in egg yolk like: lecithin, phospholipids and xantophylls. The said polar lipids were believed to be the component of the third eluate. Tests for Lipids
Figure 1.0 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 (Acroleins) odor Glycerol (Krauts) 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, the glycerol is dehydrated, forming acrolein, which has an unpleasant odor. The burnt fat odor indicates the presence of glycerol. In the groups experiment, the third eluate produced the burnt fat odor. According to the groups found references, it is the second eluate (cholesterol-containing) that should have the positive result. Suspected errors e.g contamination may have contributed to the erroneous result of this particular experiment. Krauts Test The group was unable to perform this test due to the unavailability of Kauts reagent. Liebermann-Burchard Test This test is used for the detection of cholesterol. A deep green color would indicate a positive result. 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. In the experiment, it is the second eluate (cholesterol-containing) that gave the positive results. Test for Lipid Unsaturation with Br2
Figure 3.0 Test for Unsaturation of Lipid

Test for Ester This test determines the presence of an ester functional group in a solution. This test is performed to analyse whether the lipid is saponifiable or non-saponifiable. 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. A burgundy colored solution indicates a positive result. The third eluate is the only one that yielded a positive result.

Eluate 1st 2nd 3rd Coconut Oil Canola Oil

Number of Br2 drops 55 67 64 24 18

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