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ISOLATION, GENERAL TEST AND HYDROLYSIS OF POLYSACCHARIDES

Frances Domini Angelica Paglinawan, Rhemcee Pascual, Juliefer May Pleos, Jan Thereese Salas, Shannen Mitchell Suga Group 7 2B Pharmacy Biochemistry Laboratory

ABSTRACT
Carbohydrates are large biological molecules, or macromolecules, each consisting of carbon (C), hydrogen (H), and oxygen (O) atoms. Carbohydrates are the most abundant class of organic compounds found in living organisms. It is a group of organic compounds that includes sugars, starches, celluloses, and gums and serves as a major energy source. The experiment sought to isolate the polysaccharide glycogen from chicken liver and comprehend the principles and reactions involved in the general tests to determine the sample's polysaccharide content. The initial step of the experiment was the isolation of glycogen from chicken liver by heating and adding 0.1% acetic acid and 5-10 drops of ethanol. The isolated glycogen undergoes general tests for polysaccharides which are the Molisch's Test utilizing 5% naphthol in 95% ethanol, with the positive result of a purple interface, and I2 Reaction involving 0.01 M I2 with deep red coloration as the indication for a positive result. The isolate was also subjected to acid hydrolysis using concentrated hydrcholric acid, and enzymatic hydrolysis using saliva.

INTRODUCTION
Carbohydrates, also known as saccharides, are carbon compounds that contain large quantities of hydroxyl groups, and are also the most important sources of energy. They have the basic general formula Cn(H2O)n and they are the most commonly found organic compounds in living organisms. They are classified into several groups, namely monosaccharides, disaccharides, and polysaccharides, depending on the number of their monosaccharide units. Monosaccharides are further divided with regards to the number of carbons they have pentoses and hexoses. Pentoses contain five carbon atoms while hexoses contain six carbon atoms. They can also be classified as aldoses or ketoses. Aldoses contain one aldehyde group while ketoses contain one ketone group within the molecule. An oligosaccharides monosaccharide units, on the other hand, range from two to ten, all linked by glycosidic bonds (a covalent bond which binds between the hemiacetal group of a saccharide). It is different from polysaccharides because it contains multiple but few carbon atoms, whereas polysaccharides may contain up to hundreds of monosaccharide units. Oligosaccharides and polysaccharides are similar, however, in the fact that both of them can be hydrolysed by heating in a slightly acidic solution. Glycogen, the major glucose storage polymer in animals, has a highly branched structure which

permits rapid release of glucose from glycogen stores, e.g., in muscle cells during exercise. The ability to rapidly mobilize glucose is more essential to animals than to plants. Glycogen is a very compact structure that results from the coiling of the polymer chains. This compactness allows large amounts of carbon energy to be stored in a small volume, with little effect on cellular osmolarity. In this experiment, glycogen was isolated from the chicken liver via precipitation. Chicken liver is used in this experiment because it is a good source to isolate glycogen from. Since glycogen is used in movement of body structures, several other good sources from which it may be isolated are muscle tissues, beef or pork liver. The isolation of glycogen from the chicken liver is attained by using the mechanism of precipitation. By mincing, grinding, and boiling the liver, separation of the proteins from the glycogen found in the sample is elicited. The carbohydrate tests used in this experiment can be divided into two classifications based on the mechanism of action which takes place and on the reagents used in the tests. The first involves the use of dehydrating acids followed by condensation reagents. This is called the two-step analysis, which often than not yield highly coloured results. The second classification is that which makes use of copper (II) ioncontaining reagents. The copper (II) ions are

reduced to cuprous oxide copper (I) oxide by the carbohydrates present in the samples. The Molischs Test shows positive results (purple interface) for all carbohydrates, with monosaccharides reacting much faster than disaccharides and polysaccharides. The Iodine Test, on the other hand, is used to identify glycogen and starch. Polysaccharides combine with iodine to form a positive result (a blue-black colour). Acid hydrolysis is performed on the isolate using concentrated hydrochloric acid while enzymatic hydrolysis is done using saliva and is to be stored in a dialyzing bag.

3. General Tests for Polysaccharides MOLISCHS TEST Add a few drops of Molischs Reagent into 1 mL glycogen solution. Carefully pour 2 mL conc. H2SO4 down the side of a tube using a glass rod to form a purple interface. I2 REACTION Add a few drops of 0.01 M I2 into the sample solution measuring 1 mL. A red colour produced would indicate the presence of glycogen. Hydrolysis of Polysaccharides ACID HYDROLYSIS Add 5 drops concentrated HCl into 5 mL of the isolate. Cover the test tube with a marble and boil it in water bath for thirty minutes. Keep the hydrolysate for Benedicts Test. Store the hydrolysate in a refrigerator if the test cannot be performed on the same day. ENZYMATIC HYDROLYSIS Place 10 mL isolated carbohydrate in a beaker. Add 2.3 mL saliva. Allow it to stand at room temperature for about thirty minutes and take note of any changes in the hydrolysates viscosity. Pour the solution into a dialyzing bag and suspend the bag overnight in a small flask or beaker with 50 mL distilled water. Remove and discard the dialyzing bag. Concentrate the solution inside the flask using an open flame to the volume of 10 mL. Test for the presence of reducing sugar in the hydrolysate by performing Benedicts Test.

EXPERIMENTAL
A. Compounds Tested (or Samples Used) Isolation of Glycogen Chicken liver, Boiling water, 0.1% acetic acid Glycogen Precipitation by Ethanol Ethanol General Tests for Carbohydrates 5% naphthol in 95% ethanol, Concentrated H2SO4, 0.01 M I2 Hydrolysis of Polysaccharides Concentrated HCl, Saliva

B. Procedure
1. Isolation of Glycogen Weigh about 3 g of chicken liver and place it on a Petri dish. Mince the sample with the use of scissors. Transfer sample to a beaker and pour 12 mL of distilled boiling water. Stir contents with a glass rod and boil for two minutes using a hot plate to precipitate the proteins proteins. Pour the mixture into a mortar and use pestle to grind the sample thoroughly until no lumps are visible. Add 3 mL distilled water and transfer the mixture into a beaker. Heat the mixture in a boiling water bath for thirty minutes and. f necessary, add distilled water to the mixture to avoid evaporation since glycogen goes to the solution when heating. Add 3 mL 0.1% acetic acid to improve precipitation of proteins. Filter the mixture abd divinde the obtained glycogen solutions into four portions and transfer to test tubes. 2. Glycogen Precipitation by Ethanol Add five to ten drops of ethanol to 1mL glycogen solution and observe precipitation.

RESULTS AND DISCUSSIONS


Table 1. Results Polysaccharides Test Molischs Test I2 Reaction for General Tests for

Results (+) Purple interface

(+) Deep red colour

The glycogen elicited a positive result upon the addition of Molischs Reagent and concentrated sulfuric acid. The chemical test would detect all carbohydrates, and some compounds containing carbohydrates in a combined form, based on the dehydration of the carbohydrate by sulfuric acid to produce an aldehyde (either furfural or a derivative). The test reagent dehydrates pentoses to form furfural and dehydrates hexoses to form 5-hydroxymethyl furfural. The furfurals further react with -

naphthol present in the test reagent to produce a purple interface.

coloration. The helical shape of the unbranched polysaccharide would trap iodine molecules causing the red coloration.

REFERENCES
http://www2.chemistry.msu.edu/faculty/reusch/ VirtTxtJml/carbhyd.htm http://www.answers.com/topic/carbohydrate http://chemistry2.csudh.edu/rpendarvis/monosac ch.html http://www.chacha.com/question/what-is-theformula-for-glycosidic-linkage-formation http://themedicalbiochemistrypage.org/carbohyd rates.php http://answers.yahoo.com/question/index?qid=2 0080911213302AACHBrn http://www.harpercollege.edu/tmps/chm/100/dgodambe/thedisk/carbo/molisch/m olisch.htm Figure 2. Dehydration of hexoses forming . 5-hydroxymethyl furfural http://www.biosci.ohiou.edu/introbioslab/Bios170 /170_2/benedict.htm http://www.harpercollege.edu/tmps/chm/100/dgodambe/thedisk/carbo/barf/barfo ed.htm http://www.harpercollege.edu/tmps/chm/100/dgodambe/thedisk/carbo/seli/seli.ht m http://en.wikipedia.org/wiki/Ketose Figure 3. Further reaction of furfurals with . -naphthol http://www.harpercollege.edu/tmps/chm/100/dgodambe/thedisk/carbo/bial/bials.h tm http://www.slideshare.net/katealyssacaton/mucic -and-barfoeds-test http://autumnmarcita.wordpress.com/2010/10/1 1/carbohydrates/ http://himedialabs.com/TD/HTBC002.pdf Figure 4. Formation of purple interface The polysaccharide glycogen also produced a positive result for I2 Reaction. The reaction between I2 and glycogen would produce a red

Figure 1. Dehydration of pentoses forming furfural