Lab Activity 5 Lipids Alaa Baraka IUGMarch2013

Lipids Lipids are Biomolecules that contain fatty acids or a steroid nucleus. Soluble in organic solvents but not in water. Named for the Greek word lipos, which means “fat.” Extracted from cells using organic solvents.

Classification of Lipids 1.Simple Lipids A. Neutral fats - Triglycerides B. Waxes 2. Conjugated Lipids (polar lipids) A. Phospholipids - contain a phosphoric acid molecule and a fat molecule. B. Glycolipid- contain a carbohydrate and a fat molecule. C. Sulfolipids - contain a sulfate radical. D. lipoprotein 3. Derived Lipids A. Fatty acids B. Glycerol C. Cholesterol and other steroid (Vit. D) D. Vitamins A, E, K

Examples 1.Butter 2.Ghee 3.Lard 4.Tallows 5.Vegetabels oils 6.Phospholipids 7.Sphengolipids 8.Terpenoids 9.Steroids

5 Fatty Acids Fatty acids Are long-chain carboxylic acids. Typically contain carbon atoms. Are insoluble in water. Can be saturated or unsaturated.

Lab Activity: Solubility in Polar and Non-polar Solvents Emulsification Lecithin as Emulsifying Agent Rancidity Acid Value Determination

1. Solubility in Polar and Non-polar Solvents Reagents Olive oil (vegetable oil), butter (animal fat), stearic acid (saturated fatty acid), oleic acid (unsaturated fatty acid), Solvents: dilute acid and alkali solutions, cold alcohol, hot alcohol, benzene, chloroform, ether and carbon tetrachloride. Procedure 1. Add a few drops of the liquid fat or 0.1 g of the solid fat in labeled test tubes. 2. To each test tube add a 1 ml of solvent and write down your observations. 3. Repeat the experiment with a different solvent and make your observations. Reagents Olive oil (vegetable oil), butter (animal fat), stearic acid (saturated fatty acid), oleic acid (unsaturated fatty acid), Solvents: dilute acid and alkali solutions, cold alcohol, hot alcohol, benzene, chloroform, ether and carbon tetrachloride. Procedure 1. Add a few drops of the liquid fat or 0.1 g of the solid fat in labeled test tubes. 2. To each test tube add a 1 ml of solvent and write down your observations. 3. Repeat the experiment with a different solvent and make your observations. 7

2. Emulsification Reagents Neutral olive oil, 0.5 % Na 2 C0 3, Rancid olive oil Procedure 1.Shake up a drop of neutral olive oil with a little water in a test tube, the fat becomes finally divided forming an emulsion, upon standing fat separates and rises to the top. 2.To 5 ml water in a test tube add 2 to 3 drops of 0.5 percent Na 2 CO 3. Introduce a drop of neutral olive oil and shake, the emulsion is not permanent and is not so transitory. 3.Repeat step 2 using rancid olive oil. In this case the alkali combines with the free fatty acids to form soap and the soap being an emulsifying agent, it emulsifies the fat. Reagents Neutral olive oil, 0.5 % Na 2 C0 3, Rancid olive oil Procedure 1.Shake up a drop of neutral olive oil with a little water in a test tube, the fat becomes finally divided forming an emulsion, upon standing fat separates and rises to the top. 2.To 5 ml water in a test tube add 2 to 3 drops of 0.5 percent Na 2 CO 3. Introduce a drop of neutral olive oil and shake, the emulsion is not permanent and is not so transitory. 3.Repeat step 2 using rancid olive oil. In this case the alkali combines with the free fatty acids to form soap and the soap being an emulsifying agent, it emulsifies the fat. 8

3. Lecithin as Emulsifying Agent Lecithin (phosphatidyl choline) is present in nerve, brain and other tissues of animals, plants. Egg yolk contains lecithin. Lecithin has a polar head (ionic) and 2 nonpolar hydrocarbon tails (fatty acid units) which make it an excellent emulsifying agent. 9

Procedure A. Preparation of lecithin: -Place an egg yolk in a 250 ml Erlenmeyer flask, add 50 ml of ether, shake vigorously. -Filter the mixture through a dry paper into a beaker and evaporate the ether on a steam- heated sand bath or water-bath. -Cool the solution, then add about 25 ml of acetone, stir until the precipitate clumps and sticks to the beaker, then pour off the acetone. 10

-Transfer ½ of the precipitate to a test tube, add 1 ml of water and 1 ml of vegetable oil, stopper the tube and shake very vigorously. -Repeat the above step in another test tube but without lecithin. Compare the two test tubes. After 5 min -Make your comments 11

RANCIDITY What is rancidity?  Rancidity is the development of unpleasant smells in fats and oils, which are often accompanied by changes in their texture and appearance.  Two types of rancidity:Hydrolytic rancidity Oxidative rancidity(auto-oxidation)

Oxidative rancidity Hydrolytic rancidity Occursduetotheoxidationoffatty acidchains,typicallybytheadditio nofoxygenacrosstheC=Cbondin unsaturatedfattyacids. Causedbythebreakingdownofali pidintoitscomponentfattyacidsan dglycerol.C-O-CO-R+H2O→C- O-H+HO-CO-R The process proceeds by afree radical mechanismcatalysedbyligh tinthepresenceofenzymeso rmetal ions Occurs more rapidly in the presence of enzymes such as lipase, and with heat and moisture

4. Rancidity Reagents Fresh olive oil, rancid olive oil, oleic acid, litmus paper 14

Procedure Try the reaction of fresh olive oil, oleic acid to litmus paper. Repeat the test with rancid oil. What is the reaction of a fresh lipid and that of rancid lipid? 15

5. Acid Value (free fatty acids) Determination Acid value is a measure of the extent to which the glycerides have been hydrolyzed by a lipase action. Hydrolysis is accelerated by heat and light. Acid value is an index of the efficiency of oil refining during which the free fatty acids are removed and the acid value falls to very low values. Rancidity is usually accompanied by formation of free fatty acids (indication of a deterioration of oils in storage conditions) 16

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Materials Oil sample, 0.1 N KOH 1% phenolphthalein solution EtherEthanol 250 Erlenmeyer flask burette 18

Procedure 1. Mix 25 ml of ether with 25 ml of ethanol, and 1-2 drops phenolphthalein in an erlenmeyer flask 2. Neutralize the mixture with 0.1N KOH 3. Dissolve 25 g of the oil in the mixed neutral solvent. 4. Titrate with 0.1 N KOH, shaking constantly until pink color which persists for 15 sec. Calculate the acid value and the percentage of free fatty acid in the tested sample. 19

Calculations Acid value = Titration (ml of 0.1 N KOH) x 5.61 Wt. of sample used % F.F.A = Titration (ml of 0.1 N KOH) x x 100 Wt. of sample used 20