Biochemistry Lab (Lipids Principles) PDF

Notes by: Shane Marni Ligan, RPh LABORATORY EXERCISE LIPIDS Introduction Lipids are amphipathic molecules that encompass fatty acids and derivatives such as diglycerides, triglycerides, phospholipids, and cholesterol. Roles in human body:...

Notes by: Shane Marni Ligan, RPh LABORATORY EXERCISE LIPIDS Introduction Lipids are amphipathic molecules that encompass fatty acids and derivatives such as diglycerides, triglycerides, phospholipids, and cholesterol. Roles in human body: ❑ Provide energy for the body ❑ Act as chemical messenger ❑ Involved in the maintenance of body temperature ❑ Involved in membrane layer formation ❑ Involved in the formation of prostaglandins and mediation of inflammation Safety Precautions In this activity, the student will be handling different chemicals, so it is of utmost importance that safety precautions be practiced. Notes by: Shane Marni Ligan, RPh Extraction of Lipids from Egg Yolk Principle Many Phospholipids are insoluble in acetone and they precipitate out where as triglycerides, sterol, and pigments are soluble in acetone. Egg and egg yolk is very important sources of Lecithin which is a generic term to designate any group of yellow-brownish fatty substances occurring in animal and plant tissues, which are amphiphilic – they attract both water and fatty substances (and so are both hydrophilic and lipophilic), and are used for smoothing food textures, dissolving powders (emulsifying), homogenizing liquid mixtures, and repelling sticking materials. Lecithin can easily be extracted chemically using solvents such as hexane, ethanol, acetone, petroleum ether, benzene, etc., or extraction can be done mechanically. It is usually available from sources such as soybeans, eggs, milk, marine sources, rapeseed, cottonseed, and sunflower. It has low solubility in water, but is an excellent emulsifier. Notes by: Shane Marni Ligan, RPh QUALITATIVE TEST FOR LIPIDS Acrolein Test Principle Acrolein test is used to detect the presence of glycerol or fat. When fat is treated strongly in the presence of a dehydrating agent like potassium bisulphate (KHSO 4), the glycerol portion of the molecule is dehydrated to form an unsaturated aldehyde, acrolein that has a pungent irritating odor. Interpretation of Result Positive result: If glycerol present in the sample, it will give a pungent smell. Negative result: If glycerol is absent in a sample, it will not produce a pungent smell. Notes by: Shane Marni Ligan, RPh Liebermann-Burchard Test Test Objective To detect the presence of cholesterol. Principle of the Test A chemical estimation of cholesterol, the cholesterol reacts as a typical alcohol with a strong concentrated acids and the product are colored substances. Acetic anhydride is used as solvent and dehydrating agents. Interpretation of Result Positive result: It indicates cholesterol in a sample by giving bluish- green color to the solution. Negative result: The color of the solution will not change. Notes by: Shane Marni Ligan, RPh Copper Acetate Test Principle This test used to distinguish between oil or neutral fat and fatty acid saturated and unsaturated. The copper acetate solution does not react with the oils, while saturated and unsaturated fatty acids react with copper acetate to form copper salt. unsaturated fatty acids can only be extracted by petroleum ether. Interpretation of Result Unsaturated fatty acid: 2 layer appearance - upper layer color green and the lower layer color blue. Saturated fatty acid: 2 layer appearance – appearance of white blue precipitate at the bottom of the upper layer. Notes by: Shane Marni Ligan, RPh Spot/Translucent Test Principle A translucent spot test is a preliminary test for the lipids, which is characterized by a translucent and greasy spot. The lipid will not wet the filter paper, unlike water. The lipids will form a greasy or translucent spot due to their greasy texture, and penetrate the filter paper. Unlike lipids, the spot of water will disappear from the paper. Interpretation of Result Positive result: Translucent spot will appear on the filter paper. Negative result: Translucent spot will not appear on the filter paper. Notes by: Shane Marni Ligan, RPh Test for Unsaturation Principle Unsaturation test is used to detect the unsaturated fatty acids or double bond in a lipid sample. All the neutral fat contains glycerides of fatty acids. Double bonds are found in the structure of unsaturated fatty acids, which becomes saturated by taking up either bromine or iodine. If the lipid contains more unsaturated fatty acids or more double bonds, it will take more iodine. Interpretation of Result Positive result: Pink color will disappear by the addition of unsaturated fatty acids. Negative result: Pink color will not disappear. Notes by: Shane Marni Ligan, RPh Materials Reagents 1. Beaker 1. Clarified butter 2. Test Tubes 2. Potassium bisulphate 3. Test Tube Rack 3. Cholesterol 4. Test Tube Holder 4. Chloroform 5. Filter Paper 5. Acetic anhydride 6. Funnel 6. Concentrated sulfuric acid 7. Petri dish 7. Oleic acid 8. Graduated cylinder 8. Petroleum ether 9. Stirring Rod 9. Vegetable oil 10. Tripod 10. Linseed oil 11. Centrifuge 11. Olive oil 12. Bunsen burner 12. Sunflower oil 13. Dropper 13. Palm oil 14. Filter paper 14. Corn oil 15. Light source 15. Coconut oil 16. Acetate 17. Ethanol 18. Copper acetate solution Extraction of Lipids from Egg Yolk Watch: https://www.youtube.com/watch?v=yuFu72d1d1I 1. Collect egg yolk from 2 egg in a beaker and mix well. 2. Add 50 ml of cold acetone and mix with glass rod and allow to stand for 15 min. 3. Collect the precipitate by centrifugation for 5 min at 4000 rpm. 4. Wash the precipitate with sufficient quantity of acetone till the supernatant is clear and colorless. 5. Extract the precipitate with about 100 ml of chloroform-methanol mixture (2:1) for 3-4 hours at room temperature. Notes by: Shane Marni Ligan, RPh 6. Filter and collect the filtrate (extract). 7. Evaporate the extract to dryness under a stream of nitrogen or at room temperature. 8. Dissolve the residue in small volume of petroleum ether (10-15 ml) and re-precipitate the phospholipid with addition of cold acetone (about 50 ml) and then allow the precipitate to settled down at the bottom after this collect the precipitate of phospholipid 9. Dry under vacuum and store in dark in a minimum volume of chloroform-methanol system or as solid. Qualitative Test for Lipids Acrolein test Watch: https://www.youtube.com/watch?v=NmphJwjowrQ&t=380s 1. Get 3 test tubes and label with clarified butter, vegetable oil and linseed oil. Add 2mL of each to their respective tube 2. Add small amount of Potassium bisulphate crystals to each tube 3. Heat the mixture directly on a burner 4. Observe the formation of a gas and odor that will be produced 5. Record this as your observation. Liebermann-Burchard Test Watch: https://www.youtube.com/watch?v=hc7cauZHNK8\ 1. Get a test tube with 2 ml of cholesterol dissolved in chloroform 2. Add 2 mL of acetic anhydride to the cholesterol solution and mix completely 3. Add 5 drops of concentrated sulfuric acid to the previous mixture by tilting the test tube and dropping on the side of the test tube then gently mix the solution 4. Observe for a bluish-green color 5. Record this as your observation. Notes by: Shane Marni Ligan, RPh Copper Acetate Test Watch: https://www.youtube.com/watch?v=f7dyyGJE6o0 1. Get 2 test tubes and label with olive oil and oleic acid. Add 2mL of each to their respective tube 2. Add petroleum ether to both test tubes 3. Shake test tubes to mix the content 4. Add copper acetate solution to both test tubes 5. Shake vigorously both test tubes 6. Observe for blue-green color 7. Record this as your observation. Spot/Translucent Test Watch: https://www.youtube.com/watch?v=NmphJwjowrQ&t=380s 1. Prepare a filter paper and drop the Clarified butter on it then press another filter paper on top of the other 2. Hold the filter paper against a light source 3. Then repeat the process with Vegetable oil and Linseed oil 4. Record your observations. Test for Unsaturation Watch: https://www.youtube.com/watch?v=zKZIlQm3emo&t=303s 1. Prepare 5 test tubes and label with Olive oil, Coconut oil, Corn oil, Palm oil and Sunflower oil. Add 1mL of each to their respective tube 2. Add few drops of Bromine Water to each tube one by one and make sure to replace the lid of the container 3. Shake each test tube vigorously 4. Note if the color of Bromine water disappeared. 5. Record your observation. Notes by: Shane Marni Ligan, RPh References Berroya, A. C., Cruz, M. B. – Books Atbp. Publishing Corp. Laboratory Manual in Biochemistry (3rd Edition). Go, J. C., Kamantigue, E. C., National University - Manila, College of Education, Arts and Sciences, Department of Mathematics and Sciences, College of Allied Health, & Department of Pharmacy. (n.d.). Biochemistry Laboratory Manual (1st Edition). National University - Manila. Notes by: Shane Marni Ligan, RPh