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

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

Abstract

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.

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

lipids is provision of an efficient energy source. Fat provides 9 calories of energy per gram or 2.25 times as much as either carbohydrate or protein. Carbohydrate is not stored in the body and protein stores are predominantly muscle, whose breakdown entails serious health consequences. Fat is stored as such and can be easily mobilized if needed. In primitive times survival may have been possible because of energy provided by metabolic use of stored fat. Lipids are a group of substances of diverse structures that share the common trait of being soluble in solvents such as ether or benzene. The major lipids of the body are triglycerides, which comprise a molecule of glycerol to which three fatty acids are bonded. Phospholipids are substances in which glycerol carries only two fatty acids plus phosphoric acid and an organic base such as serine. Cholesterol is a member of the family of large complex molecules generically called steroids. It has the capacity to carry one molecule of fatty acids. Cell membranes are predominantly composed of phospholipids and cholesterol. Cell membranes confer stability to cells and control entry or release of chemicals into or from the cell. Lipids serve as effective insulators and help in maintaining body temperature. Important organs such as the heart, kidneys, and reproductive organs are cushioned by fat. Nerves are protected by a sheath, a myelin that contains cholesterol, phospholipids, and other lipids. The animal organism carries a number of essential substances that catalyze chemical reactions in cells. These are called vitamins and are designated by letters. The B and C vitamins are soluble in water; the others, vitamins A, D, E, and K, are insoluble in water but soluble in fats. They are transported in lipids in the blood and stored in fat in the body. Lipids occur in tissues in a variety of physical forms, but the complex lipids are usually constituents of membranes, where they occur in close association with such compounds as proteins and polysaccharides, with which they interact by hydrophobic and van der Waals forces and perhaps by ionic bonds. Various solvents or solvent combinations have been suggested as extractants, but most lipid analysts use chloroform-methanol. Sources of lipids found in food are mostly in animals. Egg from a chicken could be. Egg yolk is a source of lecithin, an emulsifier and surfactant. Lot of lipids could be found in egg yolk although it has more water.

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. It is another solid-liquid technique in which the two phases are a solid (stationary phase) and a liquid (moving phase). The general approach is to extract total lipids from egg yolk using methanol and chloroform, then separate the lipid fractions, triglyceride, cholesterol, and phospholipids, by chromatography on silica gel. This experiment will investigate the properties of lipids present in chicken egg yolks, the lipid components present in the crude extract using column chromatography of the extracted lipids from chicken egg yolk.

Materials and Procedures The materials we have used were test tubes, beaker, stirring rod, Pasteur pipette, hot plate, iron stand and iron clamp. We started the procedures by extracting total lipids from chicken egg yolk. We added an equal amount of ethanol to the egg yolk to increase the polarity of the organic solvent, and mixed it to dehydrate and partially extract the polar lipids. We added hexane and then mixed it again and we had let it stood for 5 minutes, until two layers were formed, the fractions of polar and neutral lipids. We removed the upper polar fraction and added an equal amount of acetone to further precipitate the polar lipids from residual neutral ones, especially the cholesterol. We had collected the upper layer and transferred it into a clean beaker. After we had extracted the lipids from chicken egg yolk, we performed the column chromatography on the collected upper layer placed in the beaker (lipid extract). First, we prepared a small column by pouring a slurry of 0.5 silica gel in 4 mL of petroleum ether into a Pasteur pipette with the end tapered and plugged with glass wool. Then we poured 1 mL of lipid extract into the column, we saved the runthrough in a clean test tube. We have washed the column with 5 mL 9:1 mixture of petroleum ether:ethyl ether, and we collected the eluate in the same test tube as the run-through. 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. Lastly, we have washed the column with the last eluent, 5 mL

dichloromethane:methanol:water (1:3:1) and we collected the eluate in another test tube. We ended up the procedures by saving the different eluates.

Results and Discussion

Extraction of Total Lipids from Chicken Egg Yolk. Lipids are soluble in organic solvents, but sparingly soluble or insoluble in water. 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. Solubility of lipids is an important criterion for their extraction from source material and depends heavily on the type of lipid present, and the proportion of nonpolar (principally triacylglycerols) and polar lipids (mainly phospholipids and glycolipids) in the sample; therefore, several solvent systems might be considered, depending on the type of sample and its component. The solvents of choice are usually hexane, chloroform, methanol or chloroform, methanol or water. The simple lipids include waxes, fats, and oils. These compounds are structurally similar to each other because they consist of alcohols combined with long organic acids known as fatty acids. 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. Fats are distinguished from oils in that the former are solids and the latter, liquids. Compound lipids consist of a simple lipid and some other group, such as a phosphoric acid fragment or a nitrogen-containing alcohol. Members of this family have many important functions in biological systems. For example, one group of compound lipids, the glycolipids, occur in the membranes of brain and nerve cells. Because the compound lipids are so complex and because they occur in such small concentrations, little has been known about them until recently. One of the best known compound lipids is lecithin. The steroids are a fascinating group of compounds that includes such diverse representatives as cholic acid, a component of bile; estrone, progesterone, testosterone, and other sex hormones; vitamin D; cortisone; and cholesterol. The terpenes are another very large class of naturally-occurring compounds, many with

characteristic and pleasing aromas. The oils of camphor, menthol, lemon, orange, basil and geranium are examples of terpenes, as is natural rubber. Lipoprotiens are organic compounds composed of both protein and a lipid. There are at least four groups of lipoproteins present in plasma: Highdensity lipoproteins (HDL), low-density lipoproteins (LDL), very low density lipoproteins (VLDL), and chlyomicrons. The different densities refer to the relative amounts of lipid and protein. The higher the density, the higher the protein to lipid ratio. LDLs transport cholesterol to cells and deposit excess cholesterol in the blood vessels, which increases the risk of arteriosclerosis. HDLs, however, transport cholesterol from the tissues to the liver where it is excreted, lowering the risk of arteriosclerosis. A high HDL to total cholesterol ratio is the best indication of decreased risk of arteriosclerosis. HDL levels vary from person to person and can be influenced by such things as heredity, sex, age, and physical activity. Smoking and obesity have been shown to decrease plasma HDL levels. The composition of chicken egg yolk makes up about 33% of the liquid weight of the egg; it contains approximately 60 calories, three times the caloric content of the egg white. All of the fat soluble vitamins, (A, D, E and K) are found in the egg yolk. The composition (by weight) of the most prevalent fatty acids in egg yolk is typically as follows: Unsaturated fatty acids (Oleic acid 47 %, Linoleic acid 16 %, Palmitoleic acid 5 %, Linolenic acid 2 %), Saturated fatty acids (Palmitic acid 23 %, Stearic acid 4 %, Myristic acid 1 %). Egg yolks are one of the few foods naturally containing vitamin D. The yellow color is caused by lutein and zeaxanthin, which are yellow or orange carotenoids known as xanthophylls. Column Chromatography of Lipids. The table below (Figure1) gives us the results obtained in this experiment. With the use of the collected lipid extract from chicken egg yolk, the lipids present in the crude extract was analyzed and the first eluate was triacylglyceride, second eluate was cholesterol, and the third eluate is the lecithin. Eluates 1st eluate 2nd eluate 3rd eluate

Components Triacylglycerol Cholesterol Lecithin

Figure1. Components of Eluates

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 retention results in a variety of mechanisms including hydrogen bonding, Van der Waals' forces and also ionic bonding. The solid phase is relatively polar (normal chromatography) and the more polar the lipid, the more strongly is it adsorbed. Thus, the lipids are eluted by increasingly polar solvents. 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). The former is restricted to the fractionation of complex mixtures into two or three less complex ones, the later being adopted to analyze and quantify purified fractions. Lecithin is the most polar among the three eluates, next is cholesterol and last is triacylglycerol. The first eluate was triacylglycerol or triglyceride. The eluent was 5 mL 9:1 mixture of petroleum ether:ethyl ether. Triacylglycerols are the main components of animal and plant lipids. They are an ester of three fatty acids and glycerol. They are the most concentrated source of energy in the human body and are stored in subcutaneous fat deposits where they contribute to insulation. Fat deposits contain over 70 000 kcal of stored energy, 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. Triglycerides have lower densities than water (they float on water), and at normal room temperatures may be solid or liquid. When solid, they are called "fats" or "butters" and when liquid they are called "oils". 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, whereas 14C-acetate was utilized in synthesis of fatty acids. Ethanol activated most distinctly the synthesis of glycerol moiety as compared with the synthesis of triacylglycerol fatty acids. Ethanol activated more effectively esterification of fatty acids with formation of triacylglycerols as compared with phospholipids. Incorporation of the label into glycerol molecule occurred in response to activation of glycero-glyconeogenesis by

ethanol. The image below (Figure2) gives us the structure of triacylglyceride.

Figure2. Structure of Triacylglyceride The second eluate was cholesterol. The eluent was 5 mL 5% methanol in dichloromethane. Cholesterol is a chemical compound that is naturally produced by the body and is a combination of lipid (fat) and steroid. Cholesterol is a building block for cell membranes and for hormones like estrogen and testosterone. About 80% of the body's cholesterol is produced by the liver, while the rest comes from our diet. Cholesterol is only one of several lipids (fats) circulating in our blood stream. Its components, Triglycerides are an additional form of fat (3 fatty acids plus glycerol) circulating in the blood. Cholesterol and Triglycerides cannot dissolve in water due to being lipids, or fats. Because our blood is comprised primarily of water, for Cholesterol and Triglycerides to circulate through your blood, the Cholesterol and Triglycerides must be carried by protein packages called Apoproteins. The combination of Lipids and Apoproteins is known as Lipoprotein. Lipoproteins in turn are divided into two types: the first being Low Density Lipoprotein (LDL), which is a combination of 25% Apoproteins and 45% Cholesterol (also commonly called “bad Cholesterol”). LDL provides Cholesterol for necessary body functions, but in excess promotes potentially damaging Cholesterol accumulation in the artery walls. The second Lipoprotein type is High-density Cholesterol (HDL), comprised of 50% Apoproteins and 20% Cholesterol (generally known as “good Cholesterol”). HDL tends to help remove excess Cholesterol from your blood. Therefore, a relatively low ratio of LDL to HDL is desirable for lowering your risk for development of coronary artery disease. To calculate your Total Cholesterol Level, add HDL, plus LDL Cholesterol levels, plus 20% of the Triglycerides level. The chart in this report gives the ranges of the various Cholesterol components with desirable to dangerous ranges for each.

Figure4. Structure of Lecithin Figure3. Structure of Cholesterol The third eluate was lecithin. The eluent was 5 mL dichloromethane:methanol:water (1:3:1).

Lecithin is a lipid that consists mostly of choline, but also includes inositol, phosphorus, and linoleic acid. Lecithin helps to prevent arteriosclerosis, protects against cardiovascular disease, improves brain function, helps keep the liver and kidneys healthy, aids in thiamin and vitamin A absorption, and can even help to repair liver damage caused by alcoholism, this nutrient is essential to every living cell in the human body. One of the various functions of lecithin is to keep cholesterol in line. Its ability to emulsify oils and hold them in solution plays a major role in preventing gall stone formation. Together with bile and bile salts, it comprises the three major constituents of bile. Bile is mostly made up of fats, which lecithin keeps in liquid form in order to prevent gall stones from forming. On the other hand, cholesterol holds a delicate balance with the bile salts. If the balance is tipped on either side, the result could stone formation. By keeping cholesterol in check, lecithin helps prevent stone formation. As a component of the enzyme lecithin cholesterol acyl tranferase, the compound is said to help in the metabolism of cholesterol to it’s by products. As mentioned earlier, this substance is also called phosphatidylcholine and is an excellent source of choline. Much of the medical benefits of lecithin, particularly on high cholesterol-related conditions have been attributed to the presence of choline. The image below (Figure4) gives us the structure of lecithin.

We have concluded that the lipids extracted from the chicken egg yolk are separated based on its differences in solubility. There are factors affecting lipid solubility like chemical nature of the molecule, atomic or molecular formula weight, valence or charge or sphere of hydration, charge density and sphere of hydration. Also, in analyzing the lipids present in the crude extract using column chromatography, it is necessary to first isolate them quantitatively from nonlipid components. Extraction of lipids from source materials, such as food, animal and plant tissues, or microorganism, 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.

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