Extraction of Total Lipids from Chicken Egg Yolk and Column Chromatography of Lipids

Bialen, Mary Camille Joyce; Biscarra, Prince-Jerome; Calubad, Lareina; Cansino, Anjeanette; Chua, Norlene; Group 2 2B Pharmacy Biochemistry Laboratory

Lipid molecules include fats, waxes, and fat-soluble vitamins such as A, D, E and K. A column chromatographic procedure utilizing silica gel is described for separating lipid components of serum and lipoproteins into individual fractions containing hydrocarbons, cholesterol esters, triglycerides, cholesterol, free fatty acids and phospholipids. Egg-yolk lecithin has phospholipid classes and a composition that differ from soybean lecithin and may have unique functional properties. This experiment determined the components of each eluents. Lipids were based upon their polarity using column chromatography. The extracted lipids from chicken egg yolk were used in the column chromatography. The eluents used were 9:1 mixture of petroleum ether:ethyl ether, 5% methanol in dichloromethane and dichloromethane:methanol:water (1:3:1). The results obtained were analyzed and it showed that the the lipids are eluted by increasingly polar solvents. The lipids present in the crude extract were triacylglycerol, cholesterol and lecithin. The aim of this experiment is to understand and to determine the amounts of lipid components in chicken egg yolk. In the end of this experiment we had founded which lipid component in chicken egg yolk is more polar among all using column chromatography.

Abstract

Introduction
The existence of lipids started in the early sixties. Biophysicist Alec Bangham of the Animal Physiology Institute in Cambridge, England, made a discovery about lipids that they can put themselves together. When he placed lipids from egg yolks in water they arranged themselves into double layered circles the size of a cell, these lipid bubbles are known as liposome. David Deamer did an experiment were he took lipids and some DNA (with a intense fluorescent green dye attached) and placed them in a test tube and added a little water and when he discovered that the DNA ended up in the liposome. Polycyclic aromatic hydrocarbons (PAH) might have supplied the first cells with energy, when PAH's is exposed to light it can give off an electron and that could have supplied the cell with energy (That's what chlorophyll does for plants). Deamer also took liposome loaded with polymerase and put them into a beaker with two other molecules - a nucleotide and protease. They placed a dye in also that could slip through the liposome and would attach to RNA. They discovered that the liposome let the nucleotide in and the polymerase assembled it into RNA. The study of modern lipid chemistry began in the 17th and 18th centuries with early observations by Robert Boyle, Poulletier de la Salle, Antoine François de Fourcroy and others. The 19th century chemist, Chevreul, identified several fatty acids, suggested the name ‘cholesterine’ for the fatty substance in gallstones, coined the word ‘glycerine’, and showed that fats were comprised of glycerol and fatty acids. The 20th century brought many advances in the understanding of lipoprotein

structure and function, and explored relationships between lipoproteins and disease states. The lipids are one of four major families of biochemical compounds, the other three being carbohydrates, proteins, and nucleic acids. Biochemical compounds are organic compounds that occur in living organisms. The lipids are unique among organic and biochemical families because of the way in which they are classified. In all other families, members are categorized because of similar chemical structure and similar chemical properties. Lipids are characterized instead on the basis of a single physical property, their solubility. Lipids are hydrophobic and tend to be insoluble in water, but soluble in certain organic solvents such as benzene, chloroform and ether. They are commonly classified into three groups: simple lipids (neutral fats, triacylglycerol or triglyceride, and waxes), compound lipids (phospholipids such as lecithin, glycolipids, and lipoproteins), and derived lipids (fatty acids such as oleic acid and stearic acid, steroids such as cholesterol and oestrogen, and hydrocarbons). The lipid family contains a rather wide range of compounds that are structurally quite different from each other. Lipids, fats and oils, have borne the brunt of the blame for the degenerative diseases, the heart disease and cancer, are the major causes of death in the developed world. The negative view of lipids has obscured their essentiality for human health. Lipids are important for maintenance of human health and well-being in a number of ways. Probably the most important function of

It has the capacity to carry one molecule of fatty acids.lipids is provision of an efficient energy source.25 times as much as either carbohydrate or protein. First. but the complex lipids are usually constituents of membranes. They are transported in lipids in the blood and stored in fat in the body. 5 mL . and K. After we had extracted the lipids from chicken egg yolk. then separate the lipid fractions. and phospholipids. We had collected the upper layer and transferred it into a clean beaker. are insoluble in water but soluble in fats. Important organs such as the heart. we have washed the column with the last eluent. and mixed it to dehydrate and partially extract the polar lipids. Phospholipids are substances in which glycerol carries only two fatty acids plus phosphoric acid and an organic base such as serine. we performed the column chromatography on the collected upper layer placed in the beaker (lipid extract). We have washed the column with 5 mL 9:1 mixture of petroleum ether:ethyl ether. Fat is stored as such and can be easily mobilized if needed. Nerves are protected by a sheath. whose breakdown entails serious health consequences. the lipid components present in the crude extract using column chromatography of the extracted lipids from chicken egg yolk. These are called vitamins and are designated by letters. especially the cholesterol. Lastly. stirring rod. Pasteur pipette. a myelin that contains cholesterol. kidneys. This experiment will investigate the properties of lipids present in chicken egg yolks. D. and other lipids. phospholipids. Sources of lipids found in food are mostly in animals. In primitive times survival may have been possible because of energy provided by metabolic use of stored fat. Cell membranes confer stability to cells and control entry or release of chemicals into or from the cell. Lipids are a group of substances of diverse structures that share the common trait of being soluble in solvents such as ether or benzene. iron stand and iron clamp. which comprise a molecule of glycerol to which three fatty acids are bonded. We removed the upper polar fraction and added an equal amount of acetone to further precipitate the polar lipids from residual neutral ones. One of the most useful methods for the separation and purification of both solids and liquids when carrying out small-scale experiments is column chromatography. we saved the runthrough in a clean test tube. The major lipids of the body are triglycerides. where they occur in close association with such compounds as proteins and polysaccharides. The general approach is to extract total lipids from egg yolk using methanol and chloroform. E. We added an equal amount of ethanol to the egg yolk to increase the polarity of the organic solvent. by chromatography on silica gel. and reproductive organs are cushioned by fat. The animal organism carries a number of essential substances that catalyze chemical reactions in cells. We added hexane and then mixed it again and we had let it stood for 5 minutes. with which they interact by hydrophobic and van der Waals forces and perhaps by ionic bonds. Egg yolk is a source of lecithin. vitamins A. We have washed again the column with the second eluate (5 mL 5% methanol in dichloromethane) and then we collected again the eluate in another clean test tube. Lipids serve as effective insulators and help in maintaining body temperature. Various solvents or solvent combinations have been suggested as extractants. Then we poured 1 mL of lipid extract into the column. but most lipid analysts use chloroform-methanol. Cholesterol is a member of the family of large complex molecules generically called steroids.5 silica gel in 4 mL of petroleum ether into a Pasteur pipette with the end tapered and plugged with glass wool. Fat provides 9 calories of energy per gram or 2. We started the procedures by extracting total lipids from chicken egg yolk. The B and C vitamins are soluble in water. Lipids occur in tissues in a variety of physical forms. Carbohydrate is not stored in the body and protein stores are predominantly muscle. Lot of lipids could be found in egg yolk although it has more water. beaker. It is another solid-liquid technique in which the two phases are a solid (stationary phase) and a liquid (moving phase). Materials and Procedures The materials we have used were test tubes. Egg from a chicken could be. hot plate. and we collected the eluate in the same test tube as the run-through. the fractions of polar and neutral lipids. Cell membranes are predominantly composed of phospholipids and cholesterol. the others. until two layers were formed. we prepared a small column by pouring a slurry of 0. triglyceride. cholesterol. an emulsifier and surfactant.

We ended up the procedures by saving the different eluates. estrone. Myristic acid 1 %). The solvents of choice are usually hexane. The table below (Figure1) gives us the results obtained in this experiment. therefore. vitamin D. and other sex hormones. and the third eluate is the lecithin. All of the fat soluble vitamins. but sparingly soluble or insoluble in water. characteristic and pleasing aromas. methanol or chloroform. One of the best known compound lipids is lecithin. chloroform. Because the compound lipids are so complex and because they occur in such small concentrations. The yellow color is caused by lutein and zeaxanthin. Compound lipids consist of a simple lipid and some other group. Waxes are constructed of a single molecule each of alcohol and acid while fats and oils contain three fatty acid molecules for each alcohol molecule. several solvent systems might be considered. menthol. a component of bile. the higher the protein to lipid ratio. and cholesterol. Lipids are soluble in organic solvents. testosterone. Lipoprotiens are organic compounds composed of both protein and a lipid. orange. Solubility of lipids is an important criterion for their extraction from source material and depends heavily on the type of lipid present. D. methanol or water. With the use of the collected lipid extract from chicken egg yolk. (A. lowering the risk of arteriosclerosis. The steroids are a fascinating group of compounds that includes such diverse representatives as cholic acid. Linoleic acid 16 %. very low density lipoproteins (VLDL). LDLs transport cholesterol to cells and deposit excess cholesterol in the blood vessels. E and K) are found in the egg yolk. Saturated fatty acids (Palmitic acid 23 %. progesterone. it contains approximately 60 calories. The composition (by weight) of the most prevalent fatty acids in egg yolk is typically as follows: Unsaturated fatty acids (Oleic acid 47 %. such as a phosphoric acid fragment or a nitrogen-containing alcohol. sex. Fats are distinguished from oils in that the former are solids and the latter. HDLs. A high HDL to total cholesterol ratio is the best indication of decreased risk of arteriosclerosis. however. transport cholesterol from the tissues to the liver where it is excreted. which are yellow or orange carotenoids known as xanthophylls. second eluate was cholesterol. one group of compound lipids. age. These compounds are structurally similar to each other because they consist of alcohols combined with long organic acids known as fatty acids. basil and geranium are examples of terpenes. occur in the membranes of brain and nerve cells. The composition of chicken egg yolk makes up about 33% of the liquid weight of the egg. The different densities refer to the relative amounts of lipid and protein. and oils. little has been known about them until recently. lemon. the lipids present in the crude extract was analyzed and the first eluate was triacylglyceride. Members of this family have many important functions in biological systems.dichloromethane:methanol:water (1:3:1) and we collected the eluate in another test tube. There are at least four groups of lipoproteins present in plasma: Highdensity lipoproteins (HDL). fats. Column Chromatography of Lipids. and chlyomicrons. Smoking and obesity have been shown to decrease plasma HDL levels. as is natural rubber. Palmitoleic acid 5 %. cortisone. The existing procedures for the extraction of lipids from source material usually involve selective solvent extraction and the starting material may be subjected to drying prior to extraction. depending on the type of sample and its component. For example. which increases the risk of arteriosclerosis. Egg yolks are one of the few foods naturally containing vitamin D. low-density lipoproteins (LDL). The terpenes are another very large class of naturally-occurring compounds. many with Figure1. liquids. Eluates 1st eluate 2nd eluate 3rd eluate Components Triacylglycerol Cholesterol Lecithin Results and Discussion Extraction of Total Lipids from Chicken Egg Yolk. and physical activity. The higher the density. Components of Eluates . three times the caloric content of the egg white. The simple lipids include waxes. the glycolipids. Stearic acid 4 %. and the proportion of nonpolar (principally triacylglycerols) and polar lipids (mainly phospholipids and glycolipids) in the sample. HDL levels vary from person to person and can be influenced by such things as heredity. The oils of camphor. Linolenic acid 2 %).

Fat deposits contain over 70 000 kcal of stored energy. but in excess promotes potentially damaging Cholesterol accumulation in the artery walls. Van der Waals' forces and also ionic bonding. LDL provides Cholesterol for necessary body functions. HDL tends to help remove excess Cholesterol from your blood. Figure2. while the rest comes from our diet. The eluent was 5 mL 5% methanol in dichloromethane. They are an ester of three fatty acids and glycerol. or fats. Ethanol activated more effectively esterification of fatty acids with formation of triacylglycerols as compared with phospholipids. Structure of Triacylglyceride The second eluate was cholesterol. The former is restricted to the fractionation of complex mixtures into two or three less complex ones. comprised of 50% Apoproteins and 20% Cholesterol (generally known as “good Cholesterol”). Independent studies of biosynthesis of fatty acid and glycerol components of glycerolipids exhibited that 3H-leucine was mainly consumed in synthesis of glycerol moiety of phospholipids and triacylglycerols. Incorporation of the label into glycerol molecule occurred in response to activation of glycero-glyconeogenesis by ethanol. Its components. Ethanol activated most distinctly the synthesis of glycerol moiety as compared with the synthesis of triacylglycerol fatty acids. add HDL. the lipids are eluted by increasingly polar solvents. The second Lipoprotein type is High-density Cholesterol (HDL). This technique has a low resolution when used at low pressure (Solid Phase Extraction or SPE) but has a high resolution (high performance) when run at high pressure using a stationary phase made of fine particles (HPLC). Cholesterol is a chemical compound that is naturally produced by the body and is a combination of lipid (fat) and steroid. plus 20% of the Triglycerides level. which is a combination of 25% Apoproteins and 45% Cholesterol (also commonly called “bad Cholesterol”). a relatively low ratio of LDL to HDL is desirable for lowering your risk for development of coronary artery disease. for Cholesterol and Triglycerides to circulate through your blood. About 80% of the body's cholesterol is produced by the liver. plus LDL Cholesterol levels. When solid. they are called "fats" or "butters" and when liquid they are called "oils". next is cholesterol and last is triacylglycerol. The solid phase is relatively polar (normal chromatography) and the more polar the lipid. Triacylglycerols are the main components of animal and plant lipids. whereas 14C-acetate was utilized in synthesis of fatty acids. and at normal room temperatures may be solid or liquid. the more strongly is it adsorbed. To calculate your Total Cholesterol Level. Because our blood is comprised primarily of water. Lecithin is the most polar among the three eluates. Triglycerides have lower densities than water (they float on water). They are the most concentrated source of energy in the human body and are stored in subcutaneous fat deposits where they contribute to insulation. Cholesterol and Triglycerides cannot dissolve in water due to being lipids. but the triacylglycerol is not immediately accessible for muscle respiration because it must be broken down into its basic components for transport in the blood and then oxidized before entry into the krebs cycle. Cholesterol is a building block for cell membranes and for hormones like estrogen and testosterone. Triglycerides are an additional form of fat (3 fatty acids plus glycerol) circulating in the blood. The first eluate was triacylglycerol or triglyceride. Cholesterol is only one of several lipids (fats) circulating in our blood stream. The combination of Lipids and Apoproteins is known as Lipoprotein. Thus. The retention results in a variety of mechanisms including hydrogen bonding.Chromatography of lipids using a glass column filled with a suitable material is a common and useful method for fractionation of lipid classes either on an analytical or a semi-preparative scale. the Cholesterol and Triglycerides must be carried by protein packages called Apoproteins. Therefore. . The chart in this report gives the ranges of the various Cholesterol components with desirable to dangerous ranges for each. the later being adopted to analyze and quantify purified fractions. The image below (Figure2) gives us the structure of triacylglyceride. The eluent was 5 mL 9:1 mixture of petroleum ether:ethyl ether. Lipoproteins in turn are divided into two types: the first being Low Density Lipoprotein (LDL).

(1994).). Structure of Lecithin Figure3. There are factors affecting lipid solubility like chemical nature of the molecule. G. animal and plant tissues. Crisostomo. organic and Biochemistry (4th ed. & et. but also includes inositol.htm Discovery of new biosynthetic pathways . Inc. or microorganism. Extraction of lipids from source materials.al. By keeping cholesterol in check. A. Bile is mostly made up of fats. Manila: UST Publishing House Bettelheim.. it comprises the three major constituents of bile. If the balance is tipped on either side. M. the result could stone formation.T.. this substance is also called phosphatidylcholine and is an excellent source of choline. Laboratory Experiments for General.edu/faculty/reusch/ VirtTxtJml/lipids. J. charge density and sphere of hydration. Structure of Cholesterol The third eluate was lecithin. phosphorus. Legninger Principles of Biochemistry. improves brain function. Lecithin is a lipid that consists mostly of choline.L. New York: Reinhold Publishing Corporation Lehninger.com/research/lipids-wsd/ Chemistry of Lipids http://www2. Together with bile and bile salts. (2001). Much of the medical benefits of lecithin. should be carried out in a manner that avoids changes in the lipids or leads to the formation of artifacts it has eluates and each was differentiated by its components.A. USA: Harcourt College Publishers. valence or charge or sphere of hydration. C. (1967). Journal of Chemical Education 82(1).bookrags. & Bernaldez. particularly on high cholesterol-related conditions have been attributed to the presence of choline. (1980) A laboratory manual for biochemistry.. (2005). A. helps keep the liver and kidneys healthy. New York: W.Laboratory Manual in General Biochemistry.Quezon city: C&E Publishong. E. & Landesberg. A. Ysrael. this nutrient is essential to every living cell in the human body. in analyzing the lipids present in the crude extract using column chromatography. 103-104 Bernas. cholesterol holds a delicate balance with the bile salts. Daya. References Barreto.chemistry. One of the various functions of lecithin is to keep cholesterol in line. Quezon City: C. such as food. Chromatography. As a component of the enzyme lecithin cholesterol acyl tranferase. C. Lecithin helps to prevent arteriosclerosis. C. The image below (Figure4) gives us the structure of lecithin. University of the Philippines Book Rags: Lipid Article http://www.S. (2008).H. Also. Basic laboratory studies in biochemistry (3rd ed.M.). Heftman. On the other hand. and linoleic acid. The Biochemistry Faculty. Its ability to emulsify oils and hold them in solution plays a major role in preventing gall stone formation.msu. F. protects against cardiovascular disease. lecithin helps prevent stone formation. Lipid extraction and cholesterol quantification: A simple protocol. The eluent was 5 mL dichloromethane:methanol:water (1:3:1). We have concluded that the lipids extracted from the chicken egg yolk are separated based on its differences in solubility. aids in thiamin and vitamin A absorption. which lecithin keeps in liquid form in order to prevent gall stones from forming. Freeman.Figure4. and can even help to repair liver damage caused by alcoholism. it is necessary to first isolate them quantitatively from nonlipid components. M. the compound is said to help in the metabolism of cholesterol to it’s by products. atomic or molecular formula weight. As mentioned earlier.

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