ISOLATION OF GLYCOGEN AND QUALITATIVE ANALYSIS OF GLUCOSE, GALACTOSE, XYLOSE, FRUCTOSE, LACTOSE, SUCROSE, AND STARCH

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.

ABSTRACT

INTRODUCTION

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. [3] 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.[1] 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 -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.[4]

Furthermore, glycogen synthesized by animals, are considered as one of the most common storage forms of glucose.
[2]

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.

EXPERIMENTAL
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

isolation of glycogen. The isolated glycogen was then treated with ethanol for precipitation. 2. General Qualitative Tests for the Isolated Glycogen The isolated Glycogen was treated with Molisch's reagent. The reagent was composed of 5% -naphthol in 95% ethanol and concentrated sulfuric acid (H2SO4) for for Molisch's test and 0.01M iodine solution for iodine reaction. 3. Specific Qualitative Tests for Fructose, Lactose, Glucose, Xylose, Galactose, and Sucrose All having 0.1M concentration, fructose, lactose, glucose, xylose, galactose and sucrose were subjected to each qualitative test. The Benedict's reagent was used for the Benedict's test then follower by Barfoed's reagent for Barfoed's test, Seliwanoff's reagent for Seliwanoff's test, Orcinol reagent for Bial's Orcinol test, concentrated nitric acid for Muric test (HNO3) and Phenyhydrazine reagent for Phenylhydrazone test. The Phenyhydrazine reagent is composed of phenylhydrazine hydrochloride, sodium acetate (CH3COONa) and distilled water. The reagents were used to make the repective qualitative analysis tests. B. Procedure 1. Extraction of Glycogen from Chicken Liver Twelve mL of boiling water was poured to 3 g of minced chicken liver. The mixture was stirred and then boiled for 2 min. 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 filtered after being added with 1 mL of 0.1% CH3COOH. 2. Testing the Presence of Glycogen a. Molischs Test In test tube B, few drops of Molischs reagent (5% -naphthol in 95% ethanol) were added into a 1 mL glycogen solution. Down the side of the tube, 2 mL of concentrated H2SO4 was carefully poured to form a layer. The color at the junction of the two liquids was then observed. b. Iodine Reaction

In test tube C, few drops of 0.01M iodine were added into 1 mL glycogen solution. This test was also performed on an unknown carbohydrate. 3. Qualitative Tests for Carbohydrates Five drops of each of the carbohydrate samples glucose, galactose, xylose, fructose, lactose and sucrose was mixed with 1 mL of Benedict's reagent. The test tubes were then placed into a boiling water bath and were removed whenever the solutions give a visible result. The same procedure was applied with Barfoed's, Seliwanoff's and Bial's - Orcinol test. While in the mucic acid test of galactose and lactose, 3 drops of the carbohydrate samples were mixed with 3 drops of concentrated HNO3. The mixture was placed on a glass slide which was passed over a small flame until almost dry afterwards. And lastly, for the phenylhydrazone test of glucose, xylose, lactose, sucrose, and starch, 2 drops of the carbohydrate samples were mixed with 4 drops of freshly prepared phenylhydrazine reagent in separate test tubes. The test tubes were later heated in a boiling water bath for 30 minutes.

RESULTS AND DISCUSSIONS

When glycogen was successfully extracted from the chicken liver, it was isolated from impurities, specifically from proteins, by protein precipitation. Precipitation of the proteins, which was enhanced by 0.1% CH3COOH, was brought about by boiling the chicken liver with water. While heating, glycogen was left soluble in the solution. The precipitate was separated from the solution by the process of filtration. 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, Molischs test, iodine reaction, and Benedicts test.
Table 1. Results from testing the presence of glycogen Glycogen Precipitation Flesh precipitate (+) by Ethanol Molischs Test purple ring (+) Iodine Reaction red color (+)

Benedicts Test

brick red precipitate (+)

Molischs test is a general test for carbohydrates. Polysaccharides like glycogen are hydrolyzed by conc. H2SO4 to yield their subunits. Glucose, the subunit of glycogen, is then dehydrated with conc. H2SO4 to form 5-hydroxymethylfurfural which reacts with naphtol in 95% ethanol, the Molischs reagent, to give a purple product. The formation of a purple ring is the positive result for Molischs test. The extracted solution from the chicken liver produced this positive result. A positive iodine test indicates the presence of glycogen in a solution. Iodine reacts with glycogen to produce polyiodide chains denoted by a red color. 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. While the iodine test is used to detect the presence of unhydrolyzed glycogen, the Benedicts test can be used to detect the presence of hydrolyzed glycogen. The extracted solution from the chicken liver was tested to be positive with Benedicts test, indicating the presence of hydrolyzed glycogen or of glucosethe monosaccharide subunit of glycogen. After performing qualitative tests, namely, Benedicts, Barfoeds, Seliwanoffs, and Bials orcinol tests, on glucose, galactose, xylose, fructose, lactose, and sucrose, the following results were obtained:
Table 2. Results of the conducted qualitative tests Sugar Benedicts Test Barfoeds 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 Seliwanoffs Test faint pink (-) faint pink (-) yellow solution (-) cherry red (+) faint pink (-) cherry red (+) Bials-Orcinol Test green (-) green (-) blue green (+) reddish brown (-) green (-) green (-)

Benedict's reagent is used as a test for the presence of all monosaccharides, and generally also reducing sugars. These include glucose, galactose, mannose, lactose and maltose. Even more generally, Benedict's test will detect the presence of aldehydes (except aromatic ones), and alpha-hydroxyketones, including those that occur in certain ketoses. Thus, although the ketose fructose is not strictly a reducing sugar, it is an alphahydroxy-ketone, and gives a positive test because it is converted to the aldoses glucose and mannose by the base in the reagent.[5] Benedicts test yields a positive result with reducing sugars and is used to distinguish non-reducing and reducing ones. Any carbohydrates that contain aldehydes, hydroxymethyl ketones, or a hemiacetal are classified as reducing sugars. The Benedicts reagent consists of CuSO4, NaOH, and tartaric acid. When heated, reducing sugars can be oxidized by the Cu+2 ions of CuSO4. In other words, reducing sugars can reduce the blue Cu+2 ions to brick-red Cu2O. This brick-red precipitate is the positive result for Benedicts test and is observed only in reducing sugars. All monosaccharides are reducing sugars since they have either an aldehyde or an -hydroxymethyl ketone. Therefore, glucose, galactose, xylose, and fructose are considered reducing sugars and were tested positive with Benedicts test. Many disaccharides are also reducing sugars (if they have a hemiacetal). But since sucrose is an acetal, it is not a reducing sugar. It rather gave a negative Benedicts test even though sucrose consists of glucose and fructose which are both reducing sugars. The reason for this is that the aldehyde of glucose and -hydroxymethyl ketone of fructose are linked together in a glycosidic bond, thus there is no free reducing group available. Lactose tested positive in Benedicts test and is therefore a reducing sugar.[6] Barfoed's Test is a chemical test used for detecting the presence of monosaccharides. It is based on the reduction of copper(II) acetate to copper(I) oxide (Cu2O), which forms a brick-red precipitate. (Disaccharides may also react, but the reaction is much slower.) The aldehyde group of the monosaccharide which normally forms a cyclic hemiacetal is oxidized to the carboxylate. A number of other substances, including sodium chloride, may interfere.[7]

Seliwanoffs test bears a positive result with ketohexoses and is used to distinguish between ketohexoses and aldohexoses. The test reagent consists of resorcinol in 6M HCl. The 6M HCl dehydrates ketohexoses to 5hydroxymethylfurfural which reacts with resorcinol to produce a cherry red condensation product within 2 min. Ketoses are more rapidly dehydrated than aldoses when heated. A positive result is indicated by a cherry-red precipitate within 2 min. Fructose, a ketohexose, was tested positive while xylose, an aldopentose, was tested negative. Aldohexoses react to form the same product but with a faint pink color instead and would do so more slowly. As a result, glucose and galactose were tested negative. 6M HCl hydrolyses sucrose and lactose into their monosaccharide subunits. Sucrose, a disaccharide consisting of fructose and glucose, gave a positive test while lactose, a disaccharide consisting of glucose and galactose, gave a negative test.[8] Bials orcinol test shows a positive result for pentoses and is used to differentiate pentoses from hexoses. Bials reagent consists of orcinol, conc. HCl, and FeCl3. The conc. HCl dehydrates pentoses to form furfural which further reacts with orcinol and Fe+3 ions to produce a blue-green condensation product. A positive result for Bials orcinol test is the immediate formation of a blue-green color. Xylose, a pentose, was tested positive. When dehydrated, hexoses give 5-hydroxy-methylfufural which reacts with the reagent to yield colors such as green and reddish brown. Glucose, galactose, and fructose which are hexoses were tested negative with Bials orcinol test suggesting that they are hexoses. Lactose and sucrose which do not even have a pentose subunit were also tested negative.[9] Mucic acid test is used for galactose. Oxidation of monosaccharides by conc. Nitric acid HNO3 yields soluble dicarboxylic acids. Mucic acids are insoluble with the said oxidizing agent. A positive result for mucic acid test is the formation of an insoluble crystalline precipitate and would indicate the presence of galactose. Lactose and galactose were tested positive with mucic acid test. The phenylhydrazone test detects reducing sugars like monosaccharides and disaccharides. Phenylhydrazone reagent consists of phenylhydrazine hydrochloride and NaCH3COO. Aldoses and ketoses when they react to phenyl hydrazine, they form

crystalline product which os known as the osazones.

REFERENCES
From Books: [1] Seager, S., et. al. (2006). Chemistry for today. Australia: Thomson/Brooks/Cole. [2] Tropp, Burton E. (1997). Biochemistry : concepts and applications. Pacific Grove, CA : Brooks/Cole. From the Internet: [3] http://www.phoenix.liu.edu./~nmatsuna /che4x/e7chos.pdf. 19 February 2009. [4] http://en.wikipedia.org/wiki/Glycogen 19 February 2009. [5] http://en.wikipedia.org/wiki/Benedict%2 7s_reagent 17 February 2009 [6] http://jchemed.chem.wisc.edu/jcesoft/C CA/CCA5/MAIN/1ORGANIC/ORG18/TRAM 18/B/MENU.HTM 17 April 2001 [7] http://en.wikipedia.org/wiki/Barfoed%27 s_test 17 February 2009 [8] http://www.harpercollege.edu/tmps/chm/100/dgodambe/thedisk/carbo/se li/seli.htm 24 May 2001 [9] http://www.harpercollege.edu/tmps/chm/100/dgodambe/thedisk/carbo/bi al/bials.htm 24 May 2001