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Isolation and Characterization of Complex Lipids from Egg Yolks
Peñafuerte, Noel S., *Pimentel, Maria Danica B. Group 20 Department of Chemistry, College of Science University of Santo Tomas, España, Manila 1008
Abstract. In this experiment, the lipids found in the egg yolks of chicken egg were isolated with organic solvents. The isolated lipids are then separated into two classes: phosphorylated and non-phosphorylated with the use of acetone. The two classes of lipids solution were characterized with various chemical tests: Libermann-Burchard, Kraut’s, Salkowski, test for phosphate, ninhydrin and Molisch test. The isolation of lipid was successful however the separation into two classes is not that successful since there were a lot errors observed in the various chemical test.
INTRODUCTION Lipid ,from the Greek word “lipos” which means fat, are low molecular weight biomolecules and nonpolar chemical substances that can be extracted from plant, microbial, and animal tissues by organic solvent. It can be found in most cells and tissues, but rarely exists as free or uncombined state (Boyer, 2000). They are usually bounded to proteins and polysaccharides of tissues in complexes of widely varying degrees of stability. Because of their association with proteins and carbohydrates, it is complicated to extract and to identify the structure of lipids. Lipids have a wide variety of molecular structures and involved in various biological functions but have similar properties. Lipids are insoluble in water and ordinary solvents but soluble in organic solvents. Most lipids are ionic or polar derivatives of hydrocarbon belonging to the amphiphiles. They have ionic or polar groups which are hydrophilic and nonpolar hydrocarbons which are hydrophobic. The more polar the lipid is the stronger amphiphiles it is (Cabatit, 1988) One source of lipid is food. Food contains fats, complex lipids and steroids. Fats are triglycerides, esters of fatty acids and glycerol. Complex lipids also contain fatty acids but their
1979). 1979). 1979). Egg lipids can be divided into two general classes: non-phosphorylated and phosphorylated or phospholipids. Structure of cyclopentanoperhydrophenanthrene In this experiment the source of lipids is a chicken egg. If the compound has one or more hydroxyl groups and no carbonyl or carboxyl group then it is a sterol. The cholesterol is 84% exists as free cholesterol and 16% as cholesterol ester. Sterols are derivatives of a cyclopentanorphenanthrene nucleus or simple sterid nucleus. about 11% by weight is made up of lipids found in the egg yolk (Todd.BIOCHEMISTRY 601 LABORATORY FORMAL REPORT alcohol may be either glycerol or sphingosine. The fatty acids also contain other constituents such as phosphate choline or mono. 2001). 1977). Chicken eggs have a consistent composition of its lipids. 2 .to oligo. Figure 1. Complex lipids are usually more stable than those from simple lipids because of the ionic and polar attractions involved (Clark. On the other hand. The little variation is due to the strain and diet the chicken have. The sterid nucleus is a combination of cyclopentane and perhydrophenanthrene rings (Figure 1). The phospholipids are 65% lecithin. The egg’s lipid composition have approximately 62% if triglycerides. 20% cephalin and various minor components (Todd. 33% of phospholipids. and 5% cholesterol.saccharides (Bettelheim. if it has one carbonyl or carboxyl group then it is a steroid (Cabatit. The lipids in the egg yolk are lipoprotein in the native state (Clark. In an egg. 1988).
and B complex are also present if the feed fed to the chicken contains these vitamins. It is a sterol and is widely distributed in all cells in the body. It 3 . The chicken’s egg yolk contains saponifiable and non-saponifiable lipids. Cholesterol is an unsaturated alcohol (Figure 3). Lecithin is also known as phospatidyl choline. Saponifiable lipids are triglycerides. especially the nervous tissues. These first types of lipids are derivatives of fatty acids. waxes. and increase of salivary secretions. It is an important component of membrane lipids. They are lecithin and cholesterol.BIOCHEMISTRY 601 LABORATORY FORMAL REPORT Phospholipids contain a nitrogenous base and phosphoric acid. Its effects on the body are: decreases the blood pressure. cerebrosides. 1988). liver and nervous tissues. Egg yolk yields lecithin with arachidonic acid as one of its component fatty acids (Cabatit. Two standard lipids were used in this experiment. stearin. olein. slowing of the heart stimulation of gastric and intestinal peristalsis. cephalin. The only non-saponifiable and most abundant lipid in egg yolks is cholesterol. D. Lecithin (Figure 2) is a phospholipid that has choline as a nitrogenous base. 1978). It is present in great quantities in egg yolk. and small amounts of linoleic acid. It can be synthesized from small fragments like acetic acid. and sphingolipids which are esters that can be hydrolyzed under basic conditions. The yolks also contain inorganic substances like sodium and potassium chlorides. Lipids can be classified as saponifiable and non-saponifiable lipids. The α-lecithin is asymmetric while the β-lecithin is symmetrical. The saponifiable lipids presents are lecithin. It is also called phosphatids or phosphorized fats. Nonsaponifiable lipids such as isoprenoids and eicosanoids belong to this type because they are not esters and cannot be hydrolyzed (Seager and Slabaugh. phospholipids. 2005). It can exists in alpha or beta form. and few amounts of calcium and magnesium phosphates (EspinoCabatit. iron. Vitamins A. palmitin. sphingomyelins.
The filtrate is then placed in a separatory funnel and was extracted with equal volume of 1% NaCl solution. It was then placed in a 4 . The yolk was stirred with 80mL of a solvent mixture (CHCl3:CH3OH.BIOCHEMISTRY 601 LABORATORY FORMAL REPORT serves as an insulator in the central nervous system where it exists in its free state. Figure 2. The mixture was allowed to stand for 10 minutes. EXPERIMENTAL I. Structure of Cholesterol In this experiment. The organic layer is placed in a graduated cylinder and its volume measured. Salkowski test. The aqueous layer was discarded. v/v). The filtrate was placed in a graduated cylinder to measure its volume. The organic layer (bottom layer) is separated from the aqueous layer. Kraut’s test. the lipids in egg yolks is isolated and separated into phosphorylated and non-phosphorylated lipids. test for Phosphate. The mixture was then filtrated through a filter paper. Ninhydrin test and Molisch test. Structure of Lecithin Figure 3. 2:1. 1988). These tests are Libermann-Burchard test. The egg yolk was placed in a clean 250-mL beaker. The isolated lipids are characterized with various chemical tests. Isolation of Complex Lipids An egg was cracked and the egg yolk was separated from the egg white. (Cabatit.
The organic layer is separated and dried with anhydrous Na2SO4. The Acetone solution is carefully decanted through a filter paper and the filtrate was collected in a clean Erlenmeyer flask. The solution is transferred into a test tube and labeled as non-phosphorylated lipids (NPL). II. The acetone solution was evaporated to dryness in a water bath in the hood. The residue kept earlier is dissolved in 3mL of solvent mixture and a pinch of hydroquinone was added. Libermann-Burchard test An amount of 0. 5 . The residue is kept for used later on. The solution is transferred into a test tube and labeled as phosphorylated lipids (PL). Ten drops of acetic anhydride was added to each test tube and was gently swirled.BIOCHEMISTRY 601 LABORATORY FORMAL REPORT separatory funnel and was extracted with equal volume of 1% NaCl solution. The mixture was filtered off into a clean Erlenmeyer flask. The sticky yellow residue was added with 15mL of acetone and cooled in an ice bath for about 15 minutes. Cholesterol and lecithin also underwent the following tests to serve as standards.5mL of each of the isolated lipid and standards were placed in a separate test tube. A pinch of hydroquinone was added and transferred into an evaporating dish. A. Characterization of the Isolated Complex Lipids The isolated lipids were characterized with the following tests. The color produced was noted. The residue is dissolved in 3mL of solvent mixture and added with a pinch of hydroquinone. The solution was evaporated to dryness over a beaker of warm water in the hood. The solution was decanted and filtered. The precipitate or residue was washed with 5mL of cold acetone. Four drops of concentrated sulfuric acid (H 2SO4) was carefully added to each test tube and was mixed well.
The color of the solution and precipitate was noted.5% ammonium molybdate was added and the solution was warmed. In each of the test tube. fifteen drops of Kraut’s reagent was added.BIOCHEMISTRY 601 LABORATORY FORMAL REPORT B. Twenty drops of concentrated H2SO4 was carefully added down the side of the tubes to the solutions. The solution was heated to 65˚C. The mixture was allowed to cool and dissolved in 3mL of warm water. The test tubes were placed in a boiling water bath in the fume hood to evaporate the solvent. An amount of 3mL of 2. The test tubes were warmed for about 1 t0 2 minutes. The color of the solution and precipitate was noted. D. Salkowski test Ten drops of the lipid solutions and standards were placed separately in small test tubes. 0. Kraut’s test Ten drops of the lipids solutions and standards were placed separately into small test tubes. The solution is transferred to a test tube and acidified with 3M nitric acid. C. The dried lipid is suspended in 10 drops of distilled water. The color of the interphase was noted.5mL of the phosphorylated lipids was mixed with fusion mixture (5 times its bulk). 6 . The same procedures were done for non-phosphorylated lipid solution and the standards solutions. The mixture is ignited over a free flame until all the organic matter is burned away and the mixture turned into a grayish or colorless liquid or a white or gray ash is obtained. Test for Phosphate In a crucible.
F. This solvent can extract most of all lipids found in the egg yolk.BIOCHEMISTRY 601 LABORATORY FORMAL REPORT E. Ninhydrin test Ten drops of the lipids solution and standards were placed into separate small test tubes. Molisch test Ten drops of the lipids solution and standards were placed into separate small test tubes. The solutions were warmed for 1 to 2 minutes and the color of the solutions was notes. This can also denature proteins. The color of the interphase was noted. Lipids are also insoluble in water. 1977). Five drops of ninhydrin in ethanol was added to each of the test tube. Twenty drops of concentrated H2SO4 was slowly added to the side of the tube. and cholesterol. RESULTS AND DISCUSSION Egg yolk contains lipids such as triglycerides. This is why organic solvent can be used to extract lipids. The dried lipid is suspended in twenty drops of distilled water. The test tubes were placed in a boiling water bath in the fume hood to evaporate of the solvent. The lipids in the egg are isolated with the use of a solvent mixture which is a mixture of chloroform and methanol (2:1). The solvent mixture dissociates the lipid-protein complexes in plasma membrane in the yolk but the solvent mixture 7 . phospholipids. The solvent mixture causes the non-lipid components to transfer to the solvent system partly by ionic interactions. Some of the extracted lipids form lipid-proteins complexes (Clark. This solvent mixture is improvised by Folch’s group. The lipids are hard to isolate since they do not occur as free molecules and are covalently bonded to proteins or carbohydrates. Lipids are generally less polar than other cell constituents.
Sodium chloride is an inorganic salt. When the solvent mixture is mixed with egg yolk. 1984). Hydroquinone is first oxidized before the lipid is oxidized. non-toxic and nonflammable. some of the lipids may remain in the aqueous layer. Phospholipids are polar lipids which is why polar solvent is used to extract this. Only a small 8 . Hydroquinone is an anti-oxidant. the unsaturated fatty acid chain of lipid would react with oxygen and cleave double bonds forming aldehydes or if further exposed.BIOCHEMISTRY 601 LABORATORY FORMAL REPORT has a tendency to dissolve some non-lipid molecules such as proteins (Boyer. This inorganic solution disturbs the noncovalent bond between proteins and lipids so that the lipids will be the only one to be extracted (Switzer and Garrity. The solution is filtered off to remove the hydrated sodium sulfate. The anhydrous sodium sulfate traps and removes water molecules present in the organic layer. If single extraction was done. The solvent mixture is less dense than water which is why it is in the bottom layer. The residue is the denatured proteins while the filtrate contains the solvent mixture and the extracted lipids. it will form carboxylic acids (Scheve. The filtrate is placed in the separatory funnel which will be extracted with 1% sodium chloride solution (NaCl). It is necessary for an anti-oxidant to be added since lipids can auto-oxidize upon exposure to air or sunlight. The filtrate is added with hydroquinone. The organic layer is added with anhydrous sodium sulfate (Na 2SO4). Multiple extractions were done for more efficient extraction of lipids. 2000). If lipids are exposed to air or sunlight. it was allowed to stand and filtered off. The sodium chloride solution extracts the non-lipid components present in the filtrate. have relatively low boiling point. The solvent mixture is also chosen since it is inexpensive. 1999). Inorganic salts in aqueous solution extract the lipids that are non-covalently attached to proteins or carbohydrate. The aqueous layer consists of the water soluble components in the sample while the organic contains the lipids.
The residue left from the evaporation of the solvent is added with cold acetone. 2000). Phospholipids are more polar than nonphosphorylated lipids. The solution is filtered off. The appearances of the isolated lipid solutions obtained in the experiment.Phosphorylated Lipids (NPL) Phospholipids (PL) OBSERVATION Orange. This serves as the phospholipids solution. it may serve as impurities. It is done to make the lipid into a solution and hydroquinone was added so that the sample won’t oxidize when it is kept. The solution is then evaporated dryness in a evaporating dish in a warm water bath in the hood. This is done to remove or evaporate the solvents. Acetone is provides a mild but rapid method of dehydrating tissues and as the water content decreases the acetone extracts fats. Acetone extracts the non-phosphorylated lipids since acetone extracts non-polar and hydrophobic lipids (Boyer. it is again evaporated in the hood then added with solvent mixture and hydroquinone. sterols and other simple lipids. If too much is added. The residue contains the phospholipids while the non-phosphorylated is in the filtrate.BIOCHEMISTRY 601 LABORATORY FORMAL REPORT amount of hydroquinone is needed. The residue is mixed with solvent mixture. Acetone is used to separate the phospholipids from the non-phosphorylated lipids. Complex lipids are relatively insoluble to acetone and are converted to friable powder (Clark. This process is done under the hood since the solvent may be harmful if it is inhaled. To the filtrate. SAMPLE Non. Table 1. 1977). The appearance of the isolated lipid solution was noted (Table 1). This solution serves as the non-phosphorylated lipid solution.yellow solution Yellow solution 9 .
If cholesterol is present. test for Phosphate. In the lecithin sample. a dark brown solution is observed which is negative for this test but it indicates that water is present in the sample. This gives the intense color observed in the experiment. The concentration of cholesterol is determined by the intensity of the color. aromatic steroids and rearrangement of cholesterol molecule.BIOCHEMISTRY 601 LABORATORY FORMAL REPORT The isolated lipid solution as well as two standards. Salkowski test. a gray-violet solution is observed that is a negative result. The color begins as purplish-pink color and progresses through a light green then to a very dark green solution. In the experiment. These chemical tests are Libermann-Burchard test. The acetic anhydride is for the acetylation of the hydroxyl group of cholesterol located at c-3 then when it is reacted with concentrated sulfuric acid. Ninhydrin test and Molisch test. The color is due to the hydroxyl group (-OH) of cholesterol reacting with the acetic anhydride and concentrated sulfuric acid and the increasing conjugation of the unsaturation in the adjacent fused ring (Bettelheim. It is also known as acetic anhydride test since this tests uses acetic anhydride. In the non-phosphorylated lipid. namely cholesterol and lecithin. underwent various chemical tests to determine its properties. There is an error 10 . The Libermann-Burchard test is a specific test for the detection of cholesterol. This test is performed in the standard and sample solutions and the color is noted (Table 2). unsaturated steroids or sterol. the cholesterol produced a deep blue green solution which expected since this test is specifically for this. In the phosphorylated lipid. 1978). the test doesn’t show the deep green color instead it shows a red to dark red solution (Espino-Cabatitt. These tests are color reactions. Kraut’s test. it undergoes sulfonation and the addition of unsaturation yielding polyenes. 2001). This test is not only used as a qualitative test but also as a quantitative test. If water is present. red-violet solution is observed since some water maybe present in the sample. a deep green color will be observed.
dehydration occurs forming a bisteroid which gives a red color interphase (Espino-Cabatit. The second test performed is the Salkowski test. In the non-phosphorylated and phosphorylated lipids. In the lecithin sample. It also determines the presence of choline. both showed dark-brown interphase which indicates that they are positive for cholesterol. This test is done in all the samples and the color of interphase was noted (Table 2). only sulfuric acid was added in a chloroform solution of the samples. This taste is named after Leopold Salkowski. This reaction gives a brick red precipitate. The reagent used in this experiment is Kraut’s reagent which is bismuth subnitrate with potassium iodide in 3M nitric acid. This interphase is observed in the chloroform layer while in the acid layer a green fluorescence. It is clear that lecithin is not cholesterol. In the phosphorylated lipid. In the cholesterol standard. a red interphase was also observed. Choline with bismuth potassium iodide undergoes a complexation reaction which also gives a brick red precipitate. a red interphase is observed which is expected. This test is done in the samples and the color of the solution and its precipitate is noted (Table 2). Cholesterol is a non-phosphorylated lipid. In this test. Kraut’s test is a modification of Dragendroff’s test which is a test for alkaloids.BIOCHEMISTRY 601 LABORATORY FORMAL REPORT in this test since in the non-phosphorylated lipid solution it should be positive. Cholesterol is a non-phosphorylated lipid. This test is similar to LibermannBurchard test since it is a specific test for cholesterol. and false alkaloids. it should have been present in the non-phosphorylated lipid solution. The red interphase can also be blue. 1988). In the presence of the acid. The third test done is Kraut’s test. a red orange solution 11 . pseudo alkaloids. When Kraut’s reagent reacts with a phospholipid. In the lecithin. In the cholesterol sample. a German physiological chemist. complexation involving the phosphorylated lipid occurs. there was an error since it should have been negative. there is also an error since it should have been negative.
The solution is heated till 65˚C. The samples were mixed with fusion mixture. Both of these samples are positive for false alkaloids or cholesterol. a red orange solution is observed as well as brown precipitate. There is an error in the phosphorylated sample since cholesterol should not be present in this sample. The solution is acidified so that the ammonium hydroxide is converted to phosphate since the nitrogen is liberated. In the sample or lecithin and phosphorylated. It is a test that determines the presence of phospholipids. The color of the solutions was noted. In the sample of cholesterol and non-phosphorylated lipid. and combusted under an open flame. 1978). The cholesterol standard is a positive result. Most amino acid reacts with 12 . This is done to remove all carbon components and retain the phosphate in addition with water. This test is done on the samples and standards. primary and secondary amines. The fourth test is the test for phosphate.BIOCHEMISTRY 601 LABORATORY FORMAL REPORT and brown precipitate is observed since cholesterol is a false alkaloid. they are clear solution with black or brown precipitate. These samples are negative for the presence of phosphate group or are not phospholipids. After heating. In the lecithin sample. a red orange precipitate was observed which is negative for this test. 2. This test indicates if an amino acid is attached to the lipids.5% ammonium molybdate is added which reacts with phosphate to form ammonium phosphate molybdate which gives a light yellow to yellow green solution. In the non-phosphorylated sample and phosphorylated sample. Ninhydrin test is a chemical test that detects ammonia. The color is due to the oxidation of phosphate forming yellow to green solution depending on the concentration (Espino-Cabatit. this precipitate maybe residue of carbon. a light yellow solution is observed which is a positive test and indicates that the samples contain a phosphate group or phospholipids. The solution is also acidified with 3M nitric acid. a mixture of potassium nitrate and sodium carbonate (3:1).
1988). The color produce is purple. Molisch test is chemical test that indicates the presence of carbohydrates or sugars. a purple solution is observed which indicates the present of cephalin. This test is very sensitive and can detect the presence of carbohydrates in dilute solution as low as 0. In the non-phosphorylated lipids. a colorless solution is observed. In the lecithin test. In the non-phosphorylated and 13 . It is the hydrolysis of the glycosidic bonds present in the sample to convert them into monosaccharides then it is converted to furfurals. The reagent ninhydrin is a strong oxidizing agent and reacts with the α-amino acids. a red solution was observed which indicates that no presence of cephalin. It is named after an Austrian botanist named Hans Molisch. a purple interphase was observed which means this sample contains carbohydrates or sugars. This test was done in the sample and the color of the interphase was noted (Table 2). The solution was heated to catalyze the reactions. In the cholesterol sample. In the cholesterol sample. In the phosphorylated lipid.BIOCHEMISTRY 601 LABORATORY FORMAL REPORT ninhydrin except proline and hydroxyproline. a purple solution was observed which indicates the presence of cephalin. the interphase was colorless which indicates that cholesterol is not a carbohydrate or sugar is present. Then the furfural or its derivatives is condensed with two molecule of phenol from the Molisch reagent. The color of the solution was noted (Table 2). 1975). Molisch reagent is α-naphthol (Figure 4) dissolved in ethanol. In the lecithin sample.001% that will give a definite positive result (Espino-Cabatit. This test is only positive for cephalin because it is the only lipid with the free serine group. This indicates that cholesterol doesn’t contain cephalin. The final product would be red or purple colored interphases. This test is done on the samples and standards. It is an oxidative deamination and decarboxylation reaction of the free serine group with ninhydrin in ethanol upon heating would yield a blue violet solution (Espino-Cabatit.
This could be a positive result since the color brown is near to the color red which a positive result is. etc. The results in the various chemical tests performed in the standards and samples.g. 14 .Burchard Salkowski Kraut’s STANDARDS Cholesterol Blue-green solution Red interphase Red-orange solution and brown precipitate Clear solution and brown precipitate Colorless solution Colorless interphase Lecithin Red-violet solution Red interphase Red-orange solution and orange precipitate Light yellow solution Purple solution Purple interphase SAMPLES Nonphosphorylated Gray-violet solution Dark-brown interphase Phosphorylated Dark brown solution Dark-brown interphase Red-orange Red-orange solution and solution and brown precipitate brown precipitate Clear solution and black precipitate Red solution Brown interphase Light yellow solution Purple solution Brown interphase Phosphate Ninhydrin Molisch CONCLUSION The isolation of lipids from chicken’s egg yolk was successful. On the other hand. Figure 4 Structure of α-naphthol. The possible sources of errors are human errors. the acetone might not be cold enough). a brown interphase was observed. OBSERVATION TESTS Liebermann . technique.BIOCHEMISTRY 601 LABORATORY FORMAL REPORT phosphorylated sample. the separation into two classes was not that reliable. the reagent (e. Table 2. The two classes were not separately properly which is why some of the tests have errors. time pressure.
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