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Elissa Ira E. Garcia, Kevyn Kisses G. Gawaran, Marivel S. Herrera, Myrone Swenson A. Jayme and Robin Mae T. Jimenez Group 4 2E Medical Technology Biochemistry Laboratory
Glycogen is a common storage form of glucose. In this experiment, chicken liver was used as a sample which was extracted to get glycogen. Glycogen solution was successfully extracted from the chicken liver. Several tests were done to help prove the extraction of glycogen solution from the chicken liver. Glycogen precipitation by ethanol, Molisch's test, iodine reaction and Benedict's test gave a positive result that indicates the presence of glycogen. On the other hand, Benedict's test, Barfoed's, Seliwanoff's and Bial's - Orcinol tests confirmed that glucose and galactose is an aldohexose, xylose is an aldepentose, fructose is a ketohexose and lactose and sucrose are disaccharides.
Simple sugars are said to be the building blocks of all carbohydrates which are made up of organic compounds that have the approximate formula C(H2O)n, which accounts for the name carbohydrate (or hydrate of carbon) that is usually applied to this group of compounds. They are not truly hydrates of carbon but are polyhydroxy (alcohol) compounds that contain an aldehyde or ketone functional group. These functional groups give the carbohydrates some of their chemical properties.  Carbohydrates can be classified depending on the functional group present namely aldose which with an aldehyde functional group and a ketose which with a ketone functional group. As well as they can be classified by the number of carbons namely pentose which has 5 sides characterized by a furarose ring and hexose which has 6 sides characterized by pyranose ring. Furthermore, they can be classified by the ultimately saccharide units namely monosaccharides which are considered as the simplest, disaccharides which are composed of two monosaccharide molecules, oligosaccharides which are compose of three to ten monosaccharides and lastly polysaccharides which are compose of more than ten monosaccharides. These criteria used in classifying compounds are very important in determining how they would behave during chemical reactions. Glycogen which was extracted from the chicken liver is a polysaccharide of glucose which functions as the secondary short term energy storage in animal cells. In addition, glycogen is found in the form of granules in the cytosol in many cell types, and plays an
important role in the glucose cycle. Glycogen is a highly branched polymer that is better described as a dendrimer of about 60,000 glucose residues. Most of Glc units are linked by α-1,4 glycosidic bonds, approximately 1 in 12 Glc residues also makes alpha-1,6 glycosidic bond with a second Glc, which results in the creation of a branch. Glycogen does not possess a reducing end: the 'reducing end' glucose residue is not free but is covalently bound to a protein termed glycogenin as a beta-linkage to a surface tyrosine residue. Therefore, the many nonreducing end-branches of glycogen facilitate its rapid synthesis and catabolism.
Furthermore, glycogen synthesized by animals, are considered as one of the most common storage forms of glucose.
This experiment aims to isolate polysaccharides specifically glycogen from chicken liver and explain the principle involved in the extraction. In addition, it aims to analyze the sugars namely, glucose, galactose, xylose, fructose, lactose, sucrose, and starch, qualitatively.
A. Compounds tested and Sample Used Glucose and Galactose, Xylose, Fructose, Lactose and Sucrose, Starch and Glycogen, Chicken Liver 1. Isolation of Glycogen from Chicken Liver The chicken liver was treated with boiling water bath. It was also treated with distilled water and 0.1% acetic acid (CH3COOH) for the
Three mL of distilled water was added to the mixture which was subsequently heated in a boiling water bath for 30 min. The mixture was stirred and then boiled for 2 min. few drops of 0. Molisch’s test.01M iodine solution for iodine reaction. The reagent was composed of 5% α-naphthol in 95% ethanol and concentrated sulfuric acid (H2SO4) for for Molisch's test and 0.isolation of glycogen. for the phenylhydrazone test of glucose. Molisch’s Test In test tube B. concentrated nitric acid for Muric test (HNO3) and Phenyhydrazine reagent for Phenylhydrazone test. Table 1. xylose.01M iodine were added into 1 mL glycogen solution. Precipitation of the proteins. galactose and sucrose were subjected to each qualitative test. was brought about by boiling the chicken liver with water. Extraction of Glycogen from Chicken Liver Twelve mL of boiling water was poured to 3 g of minced chicken liver. The successful extraction of glycogen was therefore proved by acquiring positive results from the following tests which tested the presence of glycogen in the extracted solution – glycogen precipitation by ethanol. lactose. The mixture was placed on a glass slide which was passed over a small flame until almost dry afterwards. Results from testing the presence of glycogen Glycogen Precipitation Flesh precipitate (+) by Ethanol Molisch’s Test purple ring (+) Iodine Reaction red color (+) . The Benedict's reagent was used for the Benedict's test then follower by Barfoed's reagent for Barfoed's test. Xylose. Lactose. 2. The Phenyhydrazine reagent is composed of phenylhydrazine hydrochloride. The test tubes were then placed into a boiling water bath and were removed whenever the solutions give a visible result. 2. 3. glucose. fructose. Specific Qualitative Tests for Fructose. This test was also performed on an unknown carbohydrate. And lastly. RESULTS AND DISCUSSIONS When glycogen was successfully extracted from the chicken liver. by protein precipitation. Seliwanoff's and Bial's . Galactose. The isolated glycogen was then treated with ethanol for precipitation. sucrose. which was enhanced by 0.1% CH3COOH. Qualitative Tests for Carbohydrates Five drops of each of the carbohydrate samples – glucose. 3 drops of the carbohydrate samples were mixed with 3 drops of concentrated HNO3. b. Glucose. 2 drops of the carbohydrate samples were mixed with 4 drops of freshly prepared phenylhydrazine reagent in separate test tubes. B. iodine reaction. 3. specifically from proteins.Orcinol test. sodium acetate (CH3COONa) and distilled water.1M concentration.1% CH3COOH. Iodine Reaction In test tube C. few drops of Molisch’s reagent (5% α-naphthol in 95% ethanol) were added into a 1 mL glycogen solution. Testing the Presence of Glycogen a. The same procedure was applied with Barfoed's. While heating. Down the side of the tube. 2 mL of concentrated H2SO4 was carefully poured to form a layer. Procedure 1. it was isolated from impurities. xylose. and Benedict’s test. The mixture was filtered after being added with 1 mL of 0. Orcinol reagent for Bial's Orcinol test. While in the mucic acid test of galactose and lactose. fructose. galactose. glycogen was left soluble in the solution. The precipitate was separated from the solution by the process of filtration. General Qualitative Tests for the Isolated Glycogen The isolated Glycogen was treated with Molisch's reagent. The test tubes were later heated in a boiling water bath for 30 minutes. lactose. The color at the junction of the two liquids was then observed. and starch. and Sucrose All having 0. xylose. lactose and sucrose – was mixed with 1 mL of Benedict's reagent. The reagents were used to make the repective qualitative analysis tests. Seliwanoff's reagent for Seliwanoff's test.
After performing qualitative tests. This brick-red precipitate is the positive result for Benedict’s test and is observed only in reducing sugars. Barfoed’s. it is not a reducing sugar. glucose. xylose. indicating the presence of hydrolyzed glycogen or of glucose—the monosaccharide subunit of glycogen. Barfoed's Test is a chemical test used for detecting the presence of monosaccharides. While the iodine test is used to detect the presence of unhydrolyzed glycogen. and alpha-hydroxyketones. When heated. on glucose. may interfere. mannose. The extracted solution from the chicken liver produced this positive result. Any carbohydrates that contain aldehydes. the Benedict’s test can be used to detect the presence of hydrolyzed glycogen. Results of the conducted qualitative tests Sugar Benedict’s Test Barfoed’s Test brick red brick red precipitate Glucose precipitate (+) (+) brick red brick red precipitate Galactose precipitate (+) (+) brick red brick red precipitate Xylose precipitate (+) (+) brick red brick red precipitate Fructose precipitate (+) (+) brick red Lactose blue solution (-) precipitate (+) Sucrose blue solution (-) blue solution (-) Sugar Glucose Galactose Xylose Fructose Lactose Sucrose Seliwanoff’s Test faint pink (-) faint pink (-) yellow solution (-) cherry red (+) faint pink (-) cherry red (+) Bial’s-Orcinol Test green (-) green (-) blue green (+) reddish brown (-) green (-) green (-) Benedict's reagent is used as a test for the presence of all monosaccharides. Benedict’s test yields a positive result with reducing sugars and is used to distinguish non-reducing and reducing ones. fructose. Benedict’s. xylose. Many disaccharides are also reducing sugars (if they have a hemiacetal). lactose and maltose. A positive result of iodine test for glycogen is a red color which was observed when conducting the iodine test with the extracted glycogen solution. The formation of a purple ring is the positive result for Molisch’s test. But since sucrose is an acetal. The extracted solution from the chicken liver was tested to be positive with Benedict’s test. A positive iodine test indicates the presence of glycogen in a solution. Seliwanoff’s. namely. Therefore. including those that occur in certain ketoses. and fructose are considered reducing sugars and were tested positive with Benedict’s test. It rather gave a negative Benedict’s test even though sucrose consists of glucose and fructose which are both reducing sugars. H2SO4 to form 5-hydroxymethylfurfural which reacts with αnaphtol in 95% ethanol. but the reaction is much slower. . galactose. Polysaccharides like glycogen are hydrolyzed by conc. A number of other substances. reducing sugars can reduce the blue Cu+2 ions to brick-red Cu2O. to give a purple product. Thus. Glucose.Benedict’s Test brick red precipitate (+) Molisch’s test is a general test for carbohydrates.) The aldehyde group of the monosaccharide which normally forms a cyclic hemiacetal is oxidized to the carboxylate. The Benedict’s reagent consists of CuSO4. or a hemiacetal are classified as reducing sugars. NaOH. These include glucose. lactose. and generally also reducing sugars. Benedict's test will detect the presence of aldehydes (except aromatic ones). It is based on the reduction of copper(II) acetate to copper(I) oxide (Cu2O). the Molisch’s reagent. is then dehydrated with conc. and sucrose. the subunit of glycogen. αhydroxymethyl ketones. the following results were obtained: Table 2. which forms a brick-red precipitate. (Disaccharides may also react. it is an alphahydroxy-ketone. Lactose tested positive in Benedict’s test and is therefore a reducing sugar. Even more generally. reducing sugars can be oxidized by the Cu+2 ions of CuSO4. In other words. galactose. and Bial’s orcinol tests. and tartaric acid. Iodine reacts with glycogen to produce polyiodide chains denoted by a red color. All monosaccharides are reducing sugars since they have either an aldehyde or an α-hydroxymethyl ketone. galactose. and gives a positive test because it is converted to the aldoses glucose and mannose by the base in the reagent. although the ketose fructose is not strictly a reducing sugar. thus there is no free reducing group available. H2SO4 to yield their subunits. The reason for this is that the aldehyde of glucose and α-hydroxymethyl ketone of fructose are linked together in a glycosidic bond. including sodium chloride.
was tested negative. Bial’s orcinol test shows a positive result for pentoses and is used to differentiate pentoses from hexoses. Aldoses and ketoses when they react to phenyl hydrazine. A positive result is indicated by a cherry-red precipitate within 2 min. HCl.HTM 17 April 2001  http://en.wikipedia./~nmatsuna /che4x/e7chos. Mucic acids are insoluble with the said oxidizing agent. The 6M HCl dehydrates ketohexoses to 5hydroxymethylfurfural which reacts with resorcinol to produce a cherry red condensation product within 2 min. Xylose. Sucrose. was tested positive. Lactose and galactose were tested positive with mucic acid test. Pacific Grove. al. hexoses give 5-hydroxy-methylfufural which reacts with the reagent to yield colors such as green and reddish brown. (2006). When dehydrated. 19 February 2009.org/wiki/Barfoed%27 s_test 17 February 2009  http://www. Biochemistry : concepts and applications. From the Internet:  http://www.edu. 6M HCl hydrolyses sucrose and lactose into their monosaccharide subunits. CA : Brooks/Cole.  http://en.edu/tmps/chm/100/dgodambe/thedisk/carbo/bi al/bials.wisc. Mucic acid test is used for galactose. As a result. was tested positive while xylose. a ketohexose. A positive result for Bial’s orcinol test is the immediate formation of a blue-green color.wikipedia. Oxidation of monosaccharides by conc. a pentose.chem. S. The test reagent consists of resorcinol in 6M HCl.edu/jcesoft/C CA/CCA5/MAIN/1ORGANIC/ORG18/TRAM 18/B/MENU. Bial’s reagent consists of orcinol. and FeCl3.htm 24 May 2001  http://www. Lactose and sucrose which do not even have a pentose subunit were also tested negative. The phenylhydrazone test detects reducing sugars like monosaccharides and disaccharides.harpercollege. The conc.org/wiki/Benedict%2 7s_reagent 17 February 2009  http://jchemed. a disaccharide consisting of glucose and galactose. Nitric acid HNO3 yields soluble dicarboxylic acids.Seliwanoff’s test bears a positive result with ketohexoses and is used to distinguish between ketohexoses and aldohexoses. galactose.phoenix. Burton E. a disaccharide consisting of fructose and glucose. et. Fructose. (1997). gave a positive test while lactose.  http://en. they form crystalline product which os known as the osazones.edu/tmps/chm/100/dgodambe/thedisk/carbo/se li/seli. an aldopentose. A positive result for mucic acid test is the formation of an insoluble crystalline precipitate and would indicate the presence of galactose. glucose and galactose were tested negative.wikipedia.  Tropp. and fructose which are hexoses were tested negative with Bial’s orcinol test suggesting that they are hexoses.pdf. Glucose. Aldohexoses react to form the same product but with a faint pink color instead and would do so more slowly.harpercollege. Chemistry for today.liu. Australia: Thomson/Brooks/Cole.org/wiki/Glycogen 19 February 2009. Ketoses are more rapidly dehydrated than aldoses when heated.. Phenylhydrazone reagent consists of phenylhydrazine hydrochloride and NaCH3COO. conc. gave a negative test. HCl dehydrates pentoses to form furfural which further reacts with orcinol and Fe+3 ions to produce a blue-green condensation product. REFERENCES From Books:  Seager.htm 24 May 2001 .
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