Identifying Lipids Using Chemical tests

1. Acrolein Test
Principle When glycerol is heated with potassium bisulphate or concentrated !"#$% dehydration occurs and aldehyde Acrolein formed which has characteristic odour. This test responds to glycerol free or lin&ed as an ester. C
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)lycerol *aterials 1. Test compounds + #il or fat %#leic acid, !. Potassium bisulphate or conc. !"#$ Procedure

1. Place - drops of test compound in a clean and dry test tube !. Add 1 ml of conc. !"#$ carefully. #r 1.. g of / "#$ 0. eat the test tube directly. $. 1ote the characteristic pungent odour of Acrolein. !. Test of Triglycerides Principle This test can be used to distinguish between triglycerides and fatty acids. Triglycerides are chemically neutral while fatty acids are acidic due to their free carbo2ylic group and can decolori3e the al&aline red color of phenolphthalein. *aterials !.4 )lycerin in chloroform ..-4 bora2 solution 14 phenolphthalein in -.4 ethanol

Procedure 1. Place -ml of 5ora2 solution +..-4, in test tube

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Add appropriate 6olume of Phenolphthalein till red color is appearing. Add !.4 glycerin drop by drop till red color is disappearing. warm the test tube% the red color will appear again. fatty acids are not responding to this test.

0. Test of cupric acetate for detecting fatty acids This test is used for detecting saturated and unsaturated fatty acids% fatty acids reacts with cupric acetate to form cupric salts of fatty acids. The cupric salts of saturated fatty acids are not dissol6ed in water or petroleum ether7 therefore% it8s precipitated at the bottom of test tube. The cupric salts of unsaturated fatty acids are dissol6ed in petroleum ether with blue9green color. *aterials "aturated fatty acid + stearic acid or palmitic acid, Unsaturated fatty acid + #leic acid, 1.4 a:ueous cupric acetate Petroleum ether

Procedure 1. ;issol6e small mount of fatty acid in petroleum ether. !. Add appropriate amount of a:ueous cupric acetate solution. 0. "ha&e the test tube carefully and obser6e green precipitate is appear at lower a:ueous layer + cupric acetate layer, where this is happened with saturated fatty acids while at upper organic layer + petroleum ether layer, % blue or green color is appear when unsaturated fatty acid is used. $. Test for the ;egree of Unsaturation of <atty Acids +Iodine test,

<atty acids in animal fats are usually saturated% whereas those in 6egetable oils are generally unsaturated. alogens + I% 5r , will add across the double bonds and thus the decolori3ation of an iodine or bromine solution will indicate the presence of unsaturated fatty acids. Iodine test is used for distinguish between saturated and unsaturated fatty acids as well as between oils and fats. *aterials 1. #il% fat or oleic and stearic acids. !. #rganic sol6ents +chloroform or ethanol, 0. ubl8s reagent + alcoholic solution of iodine which contains some mercuric

chloride, Procedure 1. ;issol6e small amount of unsaturated fatty acid +#leic acid, in small 6olume of chloroform. !. Add ubl8s reagent drop by drop and sha&ing the tube after each addition. The halogen solution should be added =ust until it fails to be decolori3ed. 0. >ecord the number of drops needed to bring about full decolori3ation. $. >epeat the same procedure but by using saturated fatty acid + stearic acid, and Compare your results. 5. Rancidity
>ancidity is a process which is accompanied by the formation of unpleasant odour% taste% as a result of the action of moisture% air +#!, and en3ymes.To guard against rancidity% we ha6e to protect lipid from moisture and direct light as well as its storage must be in a cold place to deacti6ate lipase.

Types of rancidity:
1- Hydrolytic rancidity (or lipolytic rancidity) 2- Oxidative rancidity Kreis-Kerr test

Reagent: 1% of Phloroglucinol in alcohol or ether. Procedure 1 ml of rancid #il is mi2ed with 1 ml of conc. Cl and then add ! ml of 14 of Phloroglucinol % "ha&e and lea6e to stand for one hour. Appearance of red colour indicates the presence of aldehydes. The color intensity is increased with the increasing in the degree of rancidity in lipid.

6. Saponification of Fats and Oils

#ils can be saponified with al&ali soda to form soap and glycerol and it8s as follows?

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*aterials #il Alcoholic sodium or potassium hydro2ides+..- 1, "odium Chloride Concentrated hydrochloric acid 1. Place - g of oil or fat% 1.. g of sodium hydro2ide and !- ml of alcohol in round flas&. !. Place the boiling stone at round flas& for regulating the heating and reflu2 the solution for 0. minutes at boiling water bath. 0. Warm the solution for e6aporating the alcohol. $. Cool the solution in a bea&er of cold water. -. >emo6e a small piece of the solid product +about the si3e of a pea, using a glass stirring rod and dissol6e it with !. ml ;W and obser6e the foam formed between soap and water. @. Ammonium *olybdate Test for Phosphate The presence of free phosphate in acidic solution can be detected by adding a molybdate to the solution. A:uation illustrates the pertinent reaction between phosphate and ammonium molybdate solution in the presence of nitric acid + 1#0,.
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After a few minutes% the yellow ammonium molybdo9phosphate precipitates from the reaction mi2ture. When lipids containing phosphate groups in their structures are added to a strong acid solution such as the solution used here% the lipid hydrolyses% producing free phosphate. The free phosphate then reacts as in A:uation% forming a yellow precipitate. *aterials 1. Prepare an ice9water bath !. Place ..! g of lecithin +ege yol&, in a washed % drained test tube 0. Add 0 ml of D* nitric acid solution + 1#0,.

$. "tir the reaction mi2ture by clean glass stirring rod. -. Place the test tube containing the reaction mi2ture in ice9water bath for -91. mins. D. ;ecant the clear solution into cleaned% drained test tube and add to it 1m of ..! * ammonium molybdate solution and mi2 the reaction mi2ture using a clean glass stirring rod. @. Place the test tube containing the reaction mi2ture into a D- EC water bath for appro2imately - minutes. F. <ormation of yellow precipitate is obser6ed due to e2istence of phosphate. G. >ecord the obser6ations. F. Tests of Cholesterol Liebermann'5urchard Test for Cholesterol This test is used in the estimation of blood cholesterol.Cholesterol produces a characteristic green color when it is mi2ed with the Liebermann'5urchard reagent% a mi2ture of acetic anhydride and sulfuric acid. The change in color may be gradual% initially pin&% then blue9purple% and finally deep green. *aterial ..- 4 cholesterol in chloroform Acetic anhydride Concentrated !"#$ Procedure 1. !. 0. $. Place 1ml of ..- 4 cholesterol in chloroform in a dry test tube. Add - drops of acetic anhydride. Add 1 drop of conc. !"#$ and mi2. #bser6e the appearance of pin& color which gradually turns into deep green.

Salkowski test for cholesterol *aterial ..- 4 cholesterol in chloroform Concentrated !"#$ Procedure 1. Place 1ml of ..- 4 cholesterol in chloroform in a dry test tube. !. Add 1 ml of conc. !"#$ 0. *i2 carefully and allow to stand and separate two layers % note the red color of the upper layer +chloroform, and green fluorescence in the lower layer. Zak test for cholesterol

This test is used for determination of cholesterol in blood *aterial ..! g cholesterol in 1ml of conc. acetic acid <erric chloride Conc. Acetic acid Conc. "ulfuric acid Procedure 1. Place ..- ml of prepared cholesterol solution in a dry test tube. !. Add ! ml of colored solution + mi2ture of 1.4 ferric chloride % Conc. C Conc. !"#$, 0. #bser6e appearance deep red which refers to e2istence of cholesterol C## and