Qualitative Test for Lipid

Abstract
Upon collecting the Column Chromatography, the fourth member is tasked to use the eluate for qualitative analysis of lipids. The first eluate was from a 9:1 mixture of petroleum ether and ethyl ether, the second eluate from 5mL 5% methanol from dichloromethane and the last eluate was procured from CH 2Cl2:CH3OH:H2O (1:3:1). Two tests were performed, these are test for ester and acrolein test. Under the test for ester, both eluates numbers 1 and 3 exhibited a positive result: burgundy coloration. It is now certain that eluates 1 and 3 both contains an ester functional group. Under the acrolein test, only eluate number 2 produced the expected pungent burnt fat odor, the positive result. Eluates 1 and 3 on the other hand did not produce any noticeable odour. The molecule acrolein was responsible for the pungent odour in eluate 1, which meant it definitely had glycerol prior to the experiment. By performing these qualitative test, the group was able to characterize which eluates were saponifiable and non-saponifiable.

Introduction Carbohydrates are the active energy source of the body. Our body has a limited capability to store carbohydrates but it can convert carbohydrates into inert energy storage molecules called lipids. Our body burns these reserved energy when carbohydrates are all spent. Lipids also play another functional use by insulating the body and help maintain its temperature. 1A lipid is a fat-soluble molecule. To put it another way, lipids are insoluble in water but soluble in at least one organic solvent. The other major classes of organic compounds (nucleic acids, proteins, and carbohydrates) are much more soluble in water than in an organic solvent. The water insolubility of lipid is due to its structure, the polar groups are much smaller than its non-polar portions. 2These non-polar portions provide water repellent or hydrophobic property, hydrophobic or amphipathic small molecules that may originate entirely or in part by carbanion-based condensations of thioesters.
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waxes. Thus, the presence of the esters in glycerol is basis for saponification. There are also non-saponifiable lipids such as cholesterol.
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Acrolein test is a test for the presence of glycerin or fats. Fatty acids are further characterized based on length of fatty acid, degree of saturation or unsaturation, and ability to be synthesized in the diet.
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The yolk comprises an approximate 30% portion of the egg and contains about 4.5 grams of fat. All the fat soluble vitamins(A,D,E and K) are all found on the egg yolk. Egg yolk is a source of lecithin, an emulsifier and surfactant. The yellow color is caused by lutein and zeaxanthin which are both yellow and orange is called xanthophylls.

Methodology
Test for Ester Using the eluates obtained from the Column Chromatography. 0.5mL of Ethanol:1butanol(3:1) was added to 10 drops of the eluates in separate test tubes. 2 drops of 2M NH2OH and 3M NaOH were sequentially added, the solution was mixed well and stood for 5 minutes undisturbed. 2 drops of 6M HCl and 1 drop 5% FeCl36H2O in 0.1M HCl was added and again, the solution was mixed. Samples with esters produced a burgundy color. Acrolein Test

Unlike carbohydrates, lipids do not possess a unique functional group to characterize themselves from other compounds using 4 chemical tests. Lipids are chemically heterogenous mixtures that are further classified as saponifiable and non-saponifiable lipids. 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, examples of these are triglycerides, phospholipids, glycolipids, sphingolipids and the

A pinch amount of KHSO4 was added to 10 drops of eluates in a test tube. This test tube was heated in a boiling water bath and the odor produced was carefully observed. The burnt fat odor indicated the presence of glycerol. Results
Table 1 shows the positive results for each test

Test Ester Glycerol(Acrolein Test)

Visible results Burgundy color Burnt Fat odor

At this stage, the orthoester has a choice: reforming the carbonyl can be accompanied by expulsion of either hydroxide or the alkoxide. The former leads back to the starting materials and is unproductive (explaining why saponification is in fact an equilibrium). On the other hand, expulsion of the alkoxide generates a carboxylic acid:

Table 2 shows the visible results of the reactions of the 3 eluates toward Ester and Acrolein Tests

Eluate 1 2 3

Test for Ester Burgundy color(+) --Burgundy color(+)

Acrolein Test --Burnt fat odor(+) --The alkoxide is more basic than the conjugate base of the carboxylic acid, and hence proton transfer is rapid:

Discussion Test for ester This test determines the presence of an ester fuctional group in a solution. The group has performed this test in the laboratory to know whether the analyte lipid is saponifiable or non saponifiable. The principle behind this test is based on 1The reaction known as saponification, it is the way most of the soaps are manufactured. Refluxing an ester with aqueous NaOH produces an alcohol and the sodium salt of the acid.
O O || H2O || R C OR' + NaOH ====== R C O Na+ + R'OH

Saponification to the sodium salt of myristic acid takes place with NaOH in water. The acid itself can be obtained by adding dilute hydrochloric acid. Acrolein Test 3 Acrolein is the simplest unsaturated aldehyde. It is produced widely but is most often immediately reacted with other products due to its instability and toxicity. It has a piercing, disagreeable, acrid smell similar to that of burning fat, it can be produced by heating glycerol to 280 Celsius or in this case, by reacting potassium bisulfate with glycerol. 5The test that differentiates between cholesterol and lecithin is the acrolein reaction. When lipids containing glycerol are heated in the presence of potassium hydrogen sulfate, the glycerol is dehydrated, forming acrolein, which has an unpleasant odor. Further heating results in polymerization of acrolein, which is indicated by the slight blackening of the reaction mixture. Both the pungent smell and the black color indicate the

This reaction is essentially irreversible because carboxylate ion is inert towards nucleophilic substitution. Below is a more detailed illustration of the overall reaction of ester as it is being presented in a step by step fashion. 6 The mechanism by which esters are cleaved by base involves nucleophilic acyl substitution. The hydroxide anion adds to (or "attacks") the carbonyl group of the ester. The immediate product is an orthoester:

presence of glycerol and therefore fat and/or lecithin. Cholesterol gives a negative acrolein test. Illustrated below is the conversion of glycerol into acrolein using potassium bicarbonate. Acroleine can be characterized as a substance that produces a burnt fat odor.

References Books 1 Boyer, Rodney(2006). Concepts in Biochemistry, 3rd edition. John Wiley and Sons(Asia) Pte.Ltd 2 David, Nelson (2008)Lehninger Priciples of Biochemistry 5th Edition, W.H. Freeman & Co. 3 Ninfa, A., Ballou, D., Benore M.(2005). Fundamental Laboratory Approaches for Biochemistry and Biotechnology 2nd Ed., B1.1.1B1.1.27, John Wiley & Sons, Inc. 4 Laboratory Manual in General Biochemistry(2010), C and E Publishing, Inc. Internet resource 5 http://www.cerlabs.com/experiments/10875404 472.pdf 6 www.wikipedia.com