Isolation and Characterization of Carbohydrates
Group 10 I. Umali, N. Valenton, M. Vicente, J. Viguilla, P. Yap 2A Medical Technology Biochemistry Laboratory
The main objective in this particular experiment is to isolate polysaccharide, glycogen, from an animal source, which in this case is chicken. The extraction is done by precipitating the proteins, with the application of high amounts of heat and 0.1% acetic acid. After getting the isolate, it was tested for the presence of polysaccharides. The sugar undergoes dehydration and reacts with the Molisch’s reagent, yielding a blue violet color. This test is performed to test for the presence of carbohydrates creating a blue violet coloration at the junction. Iodine reaction is given to determine the presence of starch. In this case, the sample glycogen resulted to a reddish brown solution which indicated a negative result. Enzymatic hydrolysis was done with the use of human saliva containing amylase which hydrolyzes the alpha bonds of starch yielding glucose. Benedict’s test is performed to detect the presence of glucose, a reducing sugar, yielding a brick red precipitate as a positive result.
Simple sugars are said to be the building blocks of all carbohydrates which are made up of organic compounds that have the approximate formula Cn(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 are one of the main types of nutrients. They are the most important source of energy for your body. Carbohydrates are carbon compounds that contain large quantities of hydroxyl groups. 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. Another type of classification is as of a monosaccharide, an oligosaccharide or a polysaccharide. Anywhere from two to ten monosaccharide units, linked by glycosidic bonds, make up an oligosaccharide. Polysaccharides are much larger, containing hundreds of monosaccharide units. In a polysaccharide molecule, monosaccharide subunits are linked together by glycosidic bonds. This bond can be cleaved by hydrolysis to yield monosaccharides.
Molisch’s reaction is an indicator of the presence of carbohydrates. If carbohydrates are present a violet ring is formed by reaction with alpha-naphthol in the presence of sulfuric acid. Iodine reaction is widely used for the determination of the presence of starch and glycogen. It forms a deep red solution in the presence of glycogen, and blue in the presence of starch. This experiment aims to isolate polysaccharides from the chicken liver and explain the principle involved. It also shows the steps to perform the general tests for carbohydrates and explain each principle involved. And as the experiment is done, the difference of between enzymatic and acid hydrolysis are pointed out.
Extraction of Polysaccharides from Chicken Liver A 3 grams liver sample is cut and minced into very small pieces. Its purpose is to expose the extract from inside the liver and to maximize the amount to be extracted. 12 mL of boiling water is added to dissolve the carbohydrates. The mixture is then transferred to a beaker to be boiled for 2 minutes to precipitate the proteins. After two minutes, the mixture is homogenized using a mortar and pestle. It is placed in a water bath for another 30 minutes to further denature the proteins to form precipitates, and to dissolve remaining carbohydrates into the water. 1mL of 0.1% of acetic acid is added to
Color change is taken note of. and Benedict’s test. Hydrolysis of Polysaccharides Acid Hydrolysis 5 drops of HCl is added to 5ml of the isolate. I2 Reaction Few drops of 0. Hydrolysate Acid
Hydrolysis of Polysaccharides Viscosity Not viscous Benedict’s Test Green Solution with Brick Red precipitate Brick Red precipitate
Isolation Glycogen is successfully extracted from the chicken liver.
Iodine Reaction Benedict’s Test
Deep red color brick red precipitate (+)
General Tests for Polysaccharides
Molisch’s Test Few drops of Molisch’s reagent (5% αnaphthol in 95% ethanol) is added to 1ml of the solution. The precipitate was separated from the solution by the process of filtration. Enzymatic Hydrolysis 10ml of the isolated carbohydrate and 2. 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. to avoid disturbance to the solution. The amylase sample is extracted by rinsing the mouth with warm water for one minute. While heating. Then. Results from testing the presence of glycogen Glycogen Precipitation by Ethanol Molisch’s Test
Flesh precipitate (+) Blue violet ring (+)
. 2ml of concentrated H2SO4 is carefully transferred down through the side of the tube to form a layer. iodine reaction. Glucose.3ml of saliva is placed inside a beaker.1% CH3COOH. Concentrated sulfuric acid is introduced carefully. The mixture then is warmed in a water bath. Afterwards. Precipitation of the proteins. is then dehydrated with conc. which was enhanced by 0. the subunit of glycogen.01M I2 is added to 1ml of the sample solution. General Tests for Polysaccharides Molisch’s Test In this test. the dialyzing bag is removed and discarded. it was isolated from impurities. Afterwards. H2SO4 to form 5-
Table 1. The mixture is filtered and the isolate is divided into four for the following examinations. was brought about by boiling the chicken liver with water. glycogen was left soluble in the solution. and a purple color develops at the interface if a carbohydrate is present. it is covered by a marble to serve as a valve for pressure release and lock for the intake of contaminants in the test tube and is boiled in a water bath for 30 minutes.
Table 2.improve the precipitation of proteins. by protein denaturation that would result to precipitation. specifically from proteins. The solution is concentrated inside the flask using an open flame until it reaches the volume of 10ml. Molisch’s test. Molisch’s reagent is mixed with a dilute solution of carbohydrate. the presence of the reducing sugar is tested by performing the Benedict’s test. The color at the junction of the two liquids is observed. The solution is introduced to dialyzing bag and is suspended overnight in a small flask with 50ml distilled water. it is allowed to stand at room temperature for 30 minutes and change in the viscosity is then observed. The next day.
The acid hydrolysis is H+/H2O addition to a covalent bond so. The alpha-amylase catalyzes the rapid. only small amounts if maltose is formed. and in the case of the glucose derivative this will be a tetramethyl ether of the pyranose hemiacetal. The many glucose units in starch trap the I2 molecules and form a dark blue-black complex (7). Heating of starch in the presence of conc. All monosaccharides are reducing sugars. This compound will. In the presence of iodine. Other disaccharides such as sucrose are non-reducing sugars and will not react with Benedict's solution. The basic principle of this iodine test is that when an iodine solution (i. This is easily demonstrated by acidcatalyzed hydrolysis to the monosaccharide. the Molisch’s reagent. The glucose is formed by the relatively slow end cleavages of the oligosaccharides. maltose and isomaltose. Some disaccharides have exposed carbonyl groups and are also reducing sugars. 6 linkages.
Figure 1 solutions showing a negative result (left) and a positive result (right) in the Molisch’s test. cellulose. to give a purple product. aqueous solution of potassium iodide) comes in contact in starch. therefore it is a strongly reducing monosaccharide (12). in the case of glycogen it's the glycosidic covalent bonds that are the
target of acid hydrolysis. The formation of a purple ring is the positive result for Molisch’s test. and it constitutes a monosaccharide. These glycosidic linkages (1-4 and 1-6 carbons) are joining the monosaccharide in glycogen and their hydrolysis is quite random many oligosaccharides form in between as intermediates and eventually the result is glucose. the result showed a negative feedback. Starch is a polysaccharide that can be easily identified by the iodine test. of course. Polysaccharides are large high-molecular weight molecules constructed by joining monosaccharide units together by glycosidic bonds. a red precipitate. they all have a free reactive carbonyl group. on the sample obtained. HCl causes its hydrolysis into glucose because glucose have free aldehyde group. undergo typical aldehyde reactions. A positive result of iodine test for glycogen is a deep red color which was observed when conducting the iodine test with the extracted glycogen solution Hydrolysis of Polysaccharides Acid Hydrolysis Glycogen is a polysaccharide. If the saccharide is a reducing sugar. They are sometimes called glycans. the solution turns blue black in color. it will reduce the copper (II) ions to copper(I) oxide. regardless of molecular size. There are many polysaccharides that are resistant to or are very rapidly hydrolyzed. for example.e. The most important compounds in this class. 4 bonds.
. random hydrolysis of internal alpha-1. Benedict’s test is used as a general test for detecting reducing sugars. hydrolyze alpha-1. starch and glycogen are all polymers of glucose. Thus glycogen is initially split by alpha-amylase action into branched dextrins of medium molecular weight. Benedict’s test The Benedict's test allows us to detect the presence of reducing sugars (sugars with a free aldehyde or ketone group). The extracted solution from the chicken liver produced this positive result. Enzymatic Hydrolysis Several enzyme catalyzed hydrolyses are more specific with respect to bonds cleaved. alpha-amylase of human saliva. However. and the result pointed out that the isolate is glycogen. A positive iodine test for glycogen indicates the presence of unhydrolyzed glycoge.The reaction is shown as: C12H22O11+H+/H2O-------->2(C6H12O6) Acid hydrolysis of acetals regenerates the carbonyl and alcohol components.
Iodine Reaction An iodine test is done to test for the presence of starch.hydroxymethylfurfural which reacts with α-naphtol in 95% ethanol. amylose in starch forms a deep blue color. They do not however. Iodine reacts with glycogen to produce polyiodide chains denoted by the deep red color. nor do they hydrolyze maltoe. The final degradation products of the action of alpha-amylase on glycogen are glucose.
http://www.com/about_6584528_effe ct-benedict-solution-glucose. 2011 http://www.edu:80/faculty /reusch/VirtTxtJml/carbhyd.htm 14Date Retrieved: February 27..gov/medlineplus/carbo hydrates.htm
.elmhurst. Instead of having a purple color at the junction.htm 18Date Retrieved: February 28. David. Granner. 5th edition.html 3Date Retrieved: February 27.
Boyer. Glucose was present in the group’s enzymatic hydrolysate because it yielded a brick red precipitate which is a positive result for the Benedict’s test.org/ent ry/Hydrolysis#Hydrolysis_of_polysaccharid es 5Date Retrieved: February 27. 2011 http://www. a reducing sugar. a dark green color appeared.edu/~chm/vchemboo k/548starchiodine.harpercollege. 2011 http://www.kidslovekits. V. Since hydrolysis should have converted the glycogen to glucose.biosci.W. & Rodwell.edu/tmps/chm/100/dgodambe/thedisk/carbo/moli sch/molisch.ehow. 2011 http://www.htm 11Date Retrieved: February 27.html 17Date Retrieved: February 28. Hence. which indicates the presence of glucose.K.htm 16Date Retrieved: February 28.edu/~nsw/ench485/la b5. Principles of biochemistry. (2006). 2011 http://www2. 2011 http://www.msu. D. (2006) Cox.CHO reducing carbohydrates +2Cu2++5OHR-C O -2 carbohydrate ion +Cu2O (brick red ppt) + 3H2O
Date retrieved: February 28..nih.edu/introbioslab/Bi os170/170_2/benedict. It was evident that the group’s result in the Molisch’s test was not correct. 2011
The Benedicts test both for the acid and enzymatic hydrolysate gave a false result.R.nlm.ohiou.html 9Date Retrieved: February 27.org/car bohydrates. Harper’s Illustrated Biochemistry. 2011 http://www. Concepts in Biochemistry.html 4Date Retrieved: February 27. R. Murray. 2011 http://www. Manila: C&E publishing. 2011 http://themedicalbiochemistrypage.. R.eng.newworldencyclopedia.K. In the enzymatic hydrolysis.com/projects/starc h1/Starch%20Experiment. Nelson. a recommendation to that is that H2SO4 should be carefully added to the solution.chemistry. M. the Benedicts test should have given a brick-red precipitate.