Isolation and Characterization of Complex Lipids from Egg Yolks

Patrick Daluz, Josalline Dominguez, Yazumi Espinola*, Anna Mayleen Fermin

Department of Biological Sciences College of Science, University of Santo Tomas Espaa, Sampaloc, Manila 1055

Date Submitted: February 19, 2011

Keywords: Lipids, Phosphorylated lipid, Non phosphorylated lipid, Cholesterol, Galactocerebroside, Lecithin Abstract:
An egg yolk was used as the sample for the Isolation and Characterization of complex lipids. There were two classes of lipids that were separated in the sample: the phosphorylated lipid and the non phospholyrated lipid. The Phosphorylated lipid contained phosphate while the Non phosphorylated contained the Cholesterol. The obtained non phosphorylated and phosphorylated lipids from the sample were tested together with the given standards by the six color reaction/chemical tests.

I.

Introduction Lipids are the building blocks of fats which are insoluble in water. According to Mittal (2005), they

are two kinds of lipids; the simple and the complex lipids. Simple lipids are what we known as Cholesterol and Fatty acids, while the Complex ones are the Cholesterol esters and Glycerol esters. The Phosphorylated lipids or Phospholipids are a class belonging in the Complex lipids is known as the makers of the lipid bilayers of the cell membranes. Lecithin is a phospholipid that can be found in egg yolk (Gobley, 1847); which will be used as a standard in the Test for Phosphate and Krauts test. Other than that, the phospholipids that will be used as standards are Galactocerebroside and Sphingolipid (for the Brain samples only). The Non phosphorylated lipids are what we known as the simple lipids, for

example the Cholesterol that is found in animal cells which are essential in the body and also can be dangerous if there is too much level in the body is a non phosphorylated lipid. The core objectives of this experiment are to isolate the two classes of lipids: the Phosphorylated and Non phosphorylated lipids from the egg yolk sample. After the isolation, the characterization will be done by performing the six color reaction/chemical reaction tests (Lieberman Burchard test, Salkowski test, Test for Phosphate, Krauts test, Ninhydrin test,Molisch test) on the obtained lipid samples together with the given standards.

II. Materials For the Isolation, An egg yolk was used as the sample. The Solvent mixture was composed of CHCl3:CH3OH, 2:1, v/v. The 1% NaCl solution was used for washing. The Anhydrous Na2SO4 was used for drying. The Hydroquinone was used for the prevention of oxidation of the filtrate. The acetone was used to for the prevention of drying out. And separatory funnel was also used in the experiment. For the Characterization of Lipids, the Cholesterol, Lecithin, and Galactocerebroside were used as the standards for the six color reaction tests. Acetic anhydride and concentrated H2SO4 was used for Lieberman Burchard test. Concentrated H2SO4 was used for the Salkowski test. The Fusion mixture (composed of KNO3:Na2CO3, 3:1); 3 M HNO3 and 2.5% (NH4)2 MoO4 for the Test for Phosphate. Krauts reagent was used for Krauts test. The Molisch reagent was used for the Molisch test. And Ninhydrin solution was used for the Ninhydrin test.

III. Methodology
A. Isolation

The egg yolk was mixed with 100mL solvent mixture for 5mins. It was filtered after 10mins. Inside the separatory funnel, the filtrate was extracted with the equal volume of 1% NaCl solution. The aqueous layer was discarded, and the organic layer was washed twice with 1% NaCl solution.

The aqueous layer was again discarded, and the organic layer was dried by Anhydrous Na2SO4. It was filtered with filter paper; the filtrate was added by Hydroquinone to prevent oxidation. It was evaporated to dryness and was added by 15 ml Acetone. It was placed on an ice bath for 15 mins. to precipitate out the phosphatides; it was decanted and the lipid was filtered. The filtrate was the non phosphorylated; it was evaporated to dryness and was dissolved in 3ml CHCl3: HcOH + Hydroquinone. The residue was the phosphorylated lipid; it was washed with 5ml cold acetone and was decanted. The residue was discarded, and the filtrate was added by an acetone solution and was evaporated over a steam bath. The precipitate contained the cholesterol and was added with 3ml CHCl3: NaOH mixture and a pinch of Hydroquinone; and it was kept inside the refrigerator.

B. Characterization

In the Lieberman Burchard test, the Cholesterol was used as a standard together with the obtained non phosphorylated lipid. 0.5 mL standard or the lipid was added by 10 drops Acetic anhydride and concentrated H2SO4 and was mixed. Blue green or emerald green was the expected color for the positive result. The Cholesterol was used again as a standard together with the obtained non phosphorylated lipid for the Salkowski test. 10 drops of the standard or the lipid was added by concentrated H2SO4 dropwised. The presence of a red ring or an interphase was expected for the positive result. For the Test for Phosphate, the Lecithin was used as a standard and the obtained phosphorylated as a sample. 0.5mL of the standard or the lipid sample was placed in a crucible and was ignited in a free flame for charing. When the ash turned from black to white, it was cooled down and was dissolved by 3mL warm water. It was then transferred in a test tube and was acidified by 3M HNO3. It was heated in 65 and was added with (NH4)2 MoO4 and was warmed. Yellow precipitate or yellow

solution was the expected color for the positive result. The Lecithin was used as a standard and the obtained phosphorylated as the sample for Krauts test. 10 drops of the lipid or the standard was placed in a test tube and evaporated in a water bath

inside the fume hood. It was added 10 drops of distilled water and 15 drops of Krauts reagent. It was warmed for 1-2 mins. The presence of red precipitate was the expected color for the positive result. For the Ninhydrin test, the phospholipid was used as the sample. 10 drops of the lipid sample was added with 5 drops of Ninhydrin reagent. It was again warmed for 1-2mins. Blue violet color was the expected positive result. And lastly, for the Molisch test Galactocerebroside was used as the standard. Sphingolipid was not used because our sample was an egg yolk. 10 drops of the lipid was added with 20 drops of water, 2 drops of Molisch solution, and 20 drops of concentrated H2SO4.

IV. Results
Table 1: Results of the given standards together with the lipid samples for Color Reaction Tests Chemical Test Cholesterol Non Phosphorylated Lecithin Phosphorylated Galactocerebroside Sphingolipid

Salkowski test

Red interphase

Red interphase

Lieberman Burchard test

Emerald green

Emerald green

Test for Phosphate

Yellow solution (not enough phosphate)

Yellow solution (not enough phosphate)

Krauts test

Orange solution with red precipitate

Dark red solution

Ninhydrin test

Blue violet solution

Molisch test

Colorless solution with violet interphase

Egg sample

V. Discussions The Solvent mixture in the isolation that was composed of CHCl3:CH3OH, 2:1, v/v. was used because our lipid sample is a non polar lipid. Extracting the filtrate with 1% NaCl solution makes the filtrate splits into an aqueous layer and organic layer; it is also known as the salting out method. Acetone is used in separating non phosphorylated lipids from the phosphorylated lipids because it is non reactive solvent compared to others; the separation of lipids is good because less shrinkage will happen and dehydration will not evenly destroy the lipids structure. The purpose of the Hydroquinone in the isolation is to prevent oxidation from the filtrate. The Lieberman Burchard test is a test used to detect the presence of Cholesterol, the expected color for the positive result is Emerald green; according to Campbell (2005), the color is formed due the OH group of the Cholesterol bonded with the reagent. In the table, the color of the standard and our obtained non phosphorylated lipid from our egg yolk sample is the same, which indicates that it is positive due to the presence of Cholesterol in our lipid sample. The Salkowski test is a test for Cholesterol, red interphase or solution is the expected positive result which is the same as the results found in the table. The sulfuric acid is the one responsible for the positive color. For the Test for Phosphate, yellow precipitate is the expected color for the positive result which is the same as the result in our standard, Lecithin and the lipid sample. Ammonium molybdate is the one responsible for the positive result due to its attraction to the free phosphate in the acid solution. The Krauts test is for the presence of alkaloids and amine oxide bases, orange-red solution is the expected color for the positive result.

For the Ninhydrin test, it is used to determine the presence of a-amino acids. Blue-violet color is the expected color for the positive result as same as the lipid sample. And lastly, the Molisch test is used to test the presence of carbohydrates. The expected color of the solution is colorless with violet interphase which indicates the positive result. Galactocerebroside has carbohydrates because it is composed of ceramide with a glucose residue.

VI. Conclusions We separated the Cholesterol (Non Phosphorylated lipid) and the Phosphorylated lipid in our egg yolk sample; we also tested our lipid samples in the six color reaction/tests which resulted all as positive. We can therefore conclude that the isolation and the characterization of our obtained lipid samples is successful based on the tests being performed.

VII. References

Campbell, M. K. (2005). Biochemistry. (4th ed.). Singapore: Thomson Asia Pte Ltd. Harvey Simon, M. (2006, 04 12). Cholesterol and Lipids. Retrieved 02 18, 2011, from www.urac.org: www.urac.org Mittal, S. (2005). Coronary Heart Disease in Clinical Practice . Springerlink .

An Introduction to Cholesterol Introduction Lipids are the building blocks of the fats and fatty substances found in animals and plants. They are microscopic layered spheres of oil, which, in animals, are composed mainly of cholesterol, triglycerides, proteins (called lipoproteins), and phospholipids (molecules made up of phosphoric acid, fatty acids, and nitrogen). Lipids do not dissolve in water and are stored in the body to serve as sources of energy. Cholesterol Cholesterol is a white, powdery substance that is found in all animal cells and in animal-based foods (not in plants). In spite of its bad press, cholesterol is an essential nutrient necessary for many functions, including: text continues below
y y y y

Repairing cell membranes Manufacturing vitamin D on the skin's surface Producing hormones, such as estrogen and testosterone Possibly helping cell connections in the brain that are important for learning and memory

Regardless of these benefits, when cholesterol levels rise in the blood, they can have dangerous consequences, depending on the type of cholesterol. Although the body acquires some cholesterol through diet, about two-thirds is manufactured in the liver, its production stimulated by saturated fat. Saturated fats are found in animal products, meat, and dairy products. Saturated fats are found predominantly in animal products such as meat and dairy products, and are strongly associated with higher cholesterol levels. Tropical oils -- such as palm, coconut, and coconut butter -- are also high in saturated fats. Triglycerides Triglycerides are composed of fatty acid molecules. They are the basic chemicals contained in fats in both animals and plants. Lipoproteins Lipoproteins are protein spheres that transport cholesterol, triglyceride, or other lipid molecules through the bloodstream. Most of the information about the effects of cholesterol and triglyceride actually concerns lipoproteins. Lipoproteins are categorized into five types according to size and density. They can be further defined by whether they carry cholesterol or triglycerides. Review Date: 04/12/2006 Reviewed By: Harvey Simon, M.D., Associate Professor of Medicine, Harvard Medical School; Physician, Massachusetts General Hospital. A.D.A.M., Inc. is accredited by URAC, also known as the American Accreditation HealthCare Commission (www.urac.org).

The Lieberman-Burchard or acetic anhydride test is used for the detection of cholesterol. The formation of a green or green-blue colour after a few minutes is positive. Lieberman-Burchard is a reagent used in a colourimetric test [1] to detect cholesterol, which gives a deep green colour. This colour begins as a purplish, pink colour and progresses through to a light green then very dark green colour. The colour 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. Since this test uses acetic anhydride and sulfuric acid as reagents, caution must be exercised so as not to receive severe burns. Method : Dissolve one or two crystals of cholesterol in dry chloroform in a dry test tube. Add several drops of acetic anhydride and then 2 drops of conc.H2SO4 and mix carefully. After the reaction finished, the concentration of cholesterol can be measured using spectrophotometry.