Sorveto, Dayle Daniel G. Silvestre, Richard David Suzara, John Richard. Experiment No.

BMLS-2A February 7 2013 Dr. Nelson Villarante

Abstract: Extrating the lipids ing egg yolk and perform a qualitative test for lipids LiebermannBuchard test, Phosphate test, Acrolein test and Unsatturated fatty acid test (Br test). And getting the Rf value to TLC. We delute the egg yolk with 5 volumes of 1M NaCl, and Mix the 1mL of dilute egg yolk with 1.5mL of Isopropyl alcohol in a small test tube and cover with a cork, Add 1mL of petroeleum ether, mix well,and allow them to stand for a 4-6 minutes, and we get the upper layer and transfer it to a clean test tube. Remove 10 uL to spot on a 4x10 cm TLC plate. We apply a lipid sample by using the capillary tube. And we put it to the 400 mL beaker contains a mixture of petroleum ether/ethyl ether/acetic acid and let it stand until the mixture spread to the plate. And let it dry . Keywords: TLC plate, Lipids, Lecithin, Phospholipids, cholesterol, Egg yolk

I.

Introduction:

Lipids are broadly defined as any fat-soluble (lipophilic), Naturally-occuring molecule, such as fats, oils, waxes, cholesterol, sterols and fat soluble vitamins (such as vitamins A, D, E and K). Biological functions of lipids include energy storage, acting as structural components of cell membranes, and participating as important signaling molecules. In this exercise, students gain familiarity with two separation methods used in lipid biochemistry: extraction and thin-layer chromatography. They review the different classes found in animal fat and consider how

their molecular structures influence the separation techniques used. The general approach is to extract lipids from egg yolk using methanol and chloro form, and then separate the lipid fraction (triglyceride, cholesterol, and phospholipids) by chromatography. The fractions are analyzed by thin-layer chromatography. Objectives: 1. To know the basic concept of chromatography. 2. To familiarize with the different separation methods in the analysis of lipids.

II.

Methodology: A. Extraction of lipids in Egg yolk 1. Dilute egg yolk with 5 volumes of 1M NaCl.

6. Repeat (5) using the following standards: 0.2% solution of lecithin, cholesterol, and triglyceride(use olive oil) in chloroform. B. Extraction of lipids in milk 1. Mix 1mL of Milk with 1.5mL isopropyl alcohol in a small test tube and cover with a cork. 2. Follow procedures 2-6A C. Thin-Layer Chromatography 1. Using TLC plate (4x10cm) apply 10 uL of the lipid sample using capillary tube.

2. Mix 1 Ml of Dilute egg yolk with 1.5mL of Isopropyl alcohol in a small test tube and cover with a cork. 3. Add 1 mL of petroleum ether, mix well, and allow them to stand for a minute. 4. Remove the upper layer and transfer it to a clean test tube. 2. Develop the plates in 400-mL beaker covered with aluminum foil containing 10mL of developing solvent mixture, petroleum ether/ethyl ether/ acetic acid (75:25:1 by volume) 3. Develop the plates for about 15 minutes. 4. After drying in the hood, plates are wet using a dropper containing 10% ammonium

5. Remove 10 uL to spot on a 4x10 cm TLC plate.

sulfate in water and placed on a hot plate to char.

2. Test for Glycerol (acrolein test) a. Take 10 drops of lipid sample (Test tube 1, olive oil standard; Test tube 2; Lecithin; Test tube 3, Egg yolk lipid) and add a pinch of KHSO4 b. Heat the test tube in a boiling water bath and note the odor produced. 3. Test for Lipid unsaturation a. Take 10 drops of lipid sample (Test tube 1, Olive oil standard; Test tube 2, Lecithin; Test tube 3, Egg yolk lipid) and add 5% Bromine in dichloromethane dropwise until a reddish brown color persists. b. Record the added number of drops of 5% Br2 in Dichloromethane.

D. Qualitative test for lipids 1. Test for cholesterol (Liebermann-Burchard test) a. Take 10 drops of the lipid sample (test tube 1, cholesterol standard; Test tube 2, Lecithin; Test tube 3; Egg yolk lipid) and add 10 drops of acetic anhydride and shake. b. Place 2 drops of concentrated H2SO4 c. Mix and stand. d. Note the color.

4. Test for Phosphate a) Take 10 drops of lipid sample (Test tube 1, Lecithin; Test tube 2, Olive oil; Test tube 3,

Egg yolk lipid) to an evaporating dish. b) Add 10 drops of 5M H2SO4 and heat the mixture until the contents become brown.

d) To the colorless liquid add 1 mL water, heat for 5 minutes in a boiling water bath. e) Cool and add 1mL of 1M Ammonium Molybdate. f) Note the color.

c) Cool and add 1 drop concentrated HNO3 and heat until white fumes appear.

III. Sample

Data and Results Lieberman Burchard Test Acrolein Test No. of Br2drops(Lipid Unsaturation 6 drops Negative Phosphate Test Rf Value

Olive Oil

Lecithin

Negative

12 drops

Yellow brown 0.92 (positive)

Cholesterol

Emerald green (positive)

0.41

Egg Yolk Lipid

Emerald green (Positive)

24 drops

Yellow brown (positive)

Spot 1: 0.43

Spot 2: 0.92

IV.

Discussion:

Lieberman-Burchard The Lieberman-Burchard or acetic anhydride test is used for the detection of cholesterol. The formation of a green or greenblue color after a few minutes is positive. Lieberman-Burchard is a reagent used in a colorimetric test to detect cholesterol, which gives a deep green color. This color begins as a purplish, pink color and progresses through to a light green then very dark green color. The color is due to

the hydroxyl group (-OH) of cholesterol reacting with the reagents and increasing the conjugation of the un-saturation in the adjacent fused ring. Phosphate test The presence of free phosphate in acidic

solution can be detected by adding a molybdate to the solution. The equation below illustrates the pertinent reaction between phosphate and ammonium molybdate solution in the presence of nitric acid (HNO3).

Yellow precipitate results from the reaction in the mixture. When lipids containing phosphate groups in their structures are added to strong acid solution such as the solution used, the lipid hydrolyses, producing free phosphate, forming a yellow precipitate. Acrolein Test The principle behind the acrolein test is a specific chemical reaction. This reaction is utilized to determine the presence of glycerin in a fat. By heating the fat sample in the presence of potassium bisulfate (KHSO4), which acts as a dehydrating agent, acrolein (C3H4O, or CH2=CHICHO) is formed and can easily be detected by its odor. Whenever fat is heated in the presence of a dehydrating agent, the fat molecule will shed its glycerol in the form of the unsaturated aldehyde acrolein.

presence of potassium hydrogen sulfate, the glycerol is dehydrated, forming acrolein, which has an unpleasant odor. Further heating results polymerization of acrolein, which is indicated by the slight blackening of the reaction mixture. Both the pungent smell and the black color indicate the presence of glycerol and therefore fat and/ or lecithin. Cholesterol gives a negative acrolein test.

Unsaturated fatty acids Bromine reacts via addition across the carbon-carbon double bond. Disappearance of the characteristic bromine color (red/orange/yellow) within a short period of time.

V.

Conclusion:

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. The test that differentiates between cholesterol and lecithin is the acrolein reaction. When containing glycerol are heated In the

On the qualitative test for lipids, the egg yolk is positive to the phosphate test because of the presence phosphate group the phosphatidyl choline. In the leibermann the egg yolk is also positive.

REFERENCE BOOK: Introduction to general, organic, and Biochemistry. Bettelheim, brown, Campbell and Farell Page, 528-534 Biochemistry 4th edition By. Campbell pp 614-620 Internet: www.wikipedia.org www.biochemisylab/analysis/of/lipids.com www.slideshare.com www.scribd.com Questions: 1. Which lipids are evident in egg yolk and milk? Cholesterol 2. Which lipids most/least abundant? Phospholipids 3. Explain the relative migration of the different lipids in TLC. The eluent are egg yolk and milk. And in the TLC we did, We have lipids lecithin, cholesterol and triglyceride. Lecithin and Triglyceride migrate in the TLC which means it is present in the egg yolk. But in the milk present only cholesterol migrate. The lecithin is the highest rf value that mean it is more attach in the mobile phase we use.

4. Why proteins arent soluble in the solvent mixture?

We use Isoprophyl alcohol to extract lipids. Isoprophyl alcohol both polar and non-polar groups like the lipids (e.g lecithin) both non-polar and polar are dissolve like. But the proteins not.

5. Which lipids are positive to Acrolein test; Phosphate test; and Liebermanburchard test? Acrolein test- Triglyceride Phosphate test-Cholesterol and egg yolk lipid Liebermann Burchard testLecithin and egg yolk lipid.

I HEREBY CERTIFY THAT I MADE SUBSTANIAL CONTRIBUTION IN THIS REPORT;

RICHARD DAVID SILVESTRE

DAYLEDANIEL SORVETO

RICHARD SUZARA