uprIps INTRODUCTION Upids constitute a large heterogeneous ‘group of unrelated physiological and chemical a aos together because they are fat-Ike substances which are insoluble in water Fe Soune cra sete a ‘chloroform, ether, carbon tetrachloride, alcohol and benzene. Tey af essntal constituents of practally al pt ‘and animal cells. In the human ¥y, lipids are found mostly in the cell membranes, in the brain and in the nervous tissues. Fatty acids are constituents of li y ar ids, be they fats or oils, waxes, phospholipids, cerebrosides, ie, However, some so-called lipid derivatives like steroids do not contain the fatty Beil racked athough they possess the some ‘physical properties of fats like their solubility Fatty acids found in nature have even numbers of carbon atoms in the chain. The double bonds in unsaturated fatty acids are readily attacked by halogens to give addition products, This is the basis of the iodine number determination which is a test to detect the ‘degree of unsaturation of fats and fatty acids. The more unsaturated the fatty acid, the more double bonds it has and the more halogens it will absorb. 4 i H # | 6 eee Hy C(H2C)7—C—== C (CHa) CL HyC(Hs0— (CH on iG 4 ‘oH ue to the presence of double bonds in unsaturated faty acids, geometric isomerism leads to ‘aisand trans forms of the acid. The oleic acid structure above isthe cis form. ‘The unsaturated character of olls and their conversion to solid fats 's another example of ssition of atoms to the pi-bond. Hydrogenation Is of high commercial value as sol fats are more useful and edible. Fatty acids do not dissolve in water because they form diners, Since the partially negative (8°) oxygen and the partially positive (6°) hydrogen of —COOH are used in the dimerization, no group is available for H-bonding with water molecules. saat SEES Depart Caege of Since end infomaten Techn, Nene Ge Zamboanga University, Zamboanga City, Philippines.19 NURBIO LAB: Biochemistry ‘Laboratory Manual HC: O—on- F ig iweaal pa zed by dilute acids completely to fatty acids and glycerol; by alkalis to soap and (saponification); by enzyme lipase into a mocture of fatty acids, glycerol and glycerides. The glycerol released can be detected is by dehydration. The product formed is acrolein or Propenal which has a pungent irritating odor. i er power of soap and detergent is due to their emulsifying action and their ability to a ‘surface: tension. ‘The soap molecule contains a polar head and a non-polar tail. The non- Polar tail dissolves in the oil droplets, while the polar carboxy! group interacts with the () portion of the aqueous phase. The emulsion is stabilized by the repelling action of the o charges of the oil droplets. ee oil www) @® __ aaueous phase / wg @ © Fats develop rancid odor and taste when exposed to air at room temperature. The double bonds of unsaturated fatty acids combine with oxygen of the air to form peroxides, volatile eriehydes, Ketones and acids, responsible forthe rancid odor. The change may be catalyzed By bacteria. Steroids are derivatives of a _parent__ hydrocarbon compound cyclopentano- perhydroxyphenantrene. The most common steroid is cholesterol found in the brain, nerve Pesue and gallstones. Both 7-dehydrocholeterol and ergosterol (from ergot rye) are irradiated by ultraviolet light to vitamin D, the fist to vitamin Ds ‘and the second to vitamin D2. wiaisal Sarees Department, College of Scenice and Information “Technology, Ateneo de Zamboanga University, Zamboanga City, Philippines.PROCEDURE CAUTION: NEVER smell any reagent, test solution or product formed unless Instructed to do so! A. Solubility Tests, 2. Pipette 1 ml of the following solvents in separate stoppered vials (or test tubes): distilled water, ethyl alcohol, ether, chloroform, benzene, 5% hydrochloric acid, 5% sodium hydroxide. From a pipette or a dropper, add 1-2 drops of cottonseed Of eech vial and shake thoroughly. Racor! the time required for the oil to issoWve. 3. On different spots of a piece of coupon bond paper, place 1-2. drops of each of these mixtures: cottonseed — ethyl alcohol and cottonseed ~ ether. Be sure the mixtures are well-shaken before dropping. Allow solvents to evaporate and ‘compare the solubility ofthe oil inthe two solvents. . Tests for Unsaturation of Fatty Acids 4, To 6 drops of carbon tetrachloride, add 3 drops of oleic acid. Then add bromine water in carbon tetrachloride drop by drop into the mixture, shaking vial after ‘each addition. Note the number of drops needed to produce a faint orange color. 5. Into each of 3 test tubes or stoppered vials, place 2 mL of chloroform. Add to each test tube 0.2 g of palmitic acid, 4 drops oleic acid, and 4 drops of cottonseed oil respectively. Shake each tube thoroughly to dissolve the contents. ‘Add 4 drops of Hanus ~ iodine solution to each test tube and record the time in seconds for the color to disappear. C. Acrolein test 6. Prepare 2 test tubes, place: test tube 1-2 drops glycerol + a pinch of KHSO, test tube 2 - 2 drops cottonseed oil + pinch of KHSO, 7. Heat each tube over a low flame. Note the odor produced. aural SCeRCS Department, Colege of Scence and Information Technology, Ateneo de Zamboange University, Zamboanga City, Philippines.NURBIO LAB: Biochemistry a feboratoy ana '& Prepare 6 test tubes or vials. In each tube: 1, 2, 3 ~ place 5 drops of fresh ‘coconut ol, and each of tubes 4, 5, 6 ~ place 5 drops of rancid coconut ol. 9. Test the reaction of fresh coconut oil with 1 2 drops phenolphthalein, 1 — 2 ‘drops methyl orange and pH paper (or red and blue litmus paper if pH paper is ‘ot available). 10. Do the same reaction tests with rancid coconut oil E. Saponification 111. Weigh 1.5 g of fresh coconut oil in a dry beaker. Add 10 mL of 10% aleoholic Potassium hydroxide, Cover the beaker with a watch glass. 12. Fill the watch glass with crushed ice. Boil over a water bath until a drop of the hot solution added to cold water in a test tube does not form globules of fet. 13.Remove the watch glass to drive off the alcohol. Add 20 mL of water to the gelatinous mass and warm the solution to dissolve the soap. Use the soap Solution for the tests on the properties of soaps. F. Properties of Soap 14. Salting Out Place 10 mL of the soap solution in a beaker and add table salt gredually with stiring until no more table sat dissolves. Remove the solid soap formed from the liquid and wash it with water. Transfer a small amount of the solid soap into a test tube and shake it with distilled water to form suds. 15. Formation of Fatty Acids Place 5 mL. of the soap solution in a test tube and add 10% HCI until a precipitate forms. 16. Insoluble Soaps Place 2 test tubes, each containing 4 mL distiled water and 1 mL soap solution. To test tube 1 — add 5 drops 5% CaCh solution. To test tube 2 ~ add 5 drops 5% MgCl, solution ‘Natural Soences Department, Colepe of Scence and information Technology, Ateneo de Zamboanga Unversity, Zamboanga Cl, Phipps.NURBIO LAB: Biochemistry a ‘Manual Observe results. Repeat the test using liquid detergent instead of soap solution. Observe and compare results with the first part. G. Liebermann ~ Burchard Test for Cholesterol Place a few crystals of cholesterol in a dry evaporating dish. Add 2 ml. of chloroform ‘and 10 drops of acetic anhydride. Mix thoroughly. ‘Add 2-3 drops of concentrated sulfuric acid and shake. Note the color changes during the first few minutes. HH. Emulsfying Action of Lecithin In each of 2 test tubes, place 5 drops of 2% albumin solution. To test tube 1 - add a Pinch of cholesterol and shake the tube. To test tube 2 - add a pinch of cholesterol! ‘and 5 drops of lecithin. Shake the tube. Compare the results. © PROPER DISPOSAL: Dispose of solutions in the proper waste bottles (as acid or basic wastes, and organic or inorganic wastes). QUESTIONS Why are fatty acids insoluble in water? Explain why the cis-form is the predominant configuration of unsaturated fatty acids? What type of rancidity occurs in vegetable shortenings? How can it be prevented? Show the structure of the parent compound of cholesterol. Explain the cooperative effect of lecithin and albumin. vaene Tlotural Sences Department, College of Science end Information Technology, Ateneo de Zamboanga University, Zamboanga Cty, Phippnes.