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Gerald Calimabs, Clarence F. Carino, Nicole Kristine B. Carlos, Charis Joyce B. Cauyao, Jennifer Kierstine Chua Group 3 2GPharmacy Biochemistry Laboratory
Carbohydrates serve as the main energy source for the human body. The objective of this experiment are as follows: to isolate polysaccharides from animal and plant sources, to perform the general tests for carbohydrates, to compare the products of acid and enzymatic hydrolyses, to illustrate the specificity of alpha-amylase on the hydrolysis of the isolated polysaccharide, to prepare the dialyzing bag used in separating the products of enzymatic hydrolysis and to examine microscopically the different osazone and mucic acid crystals. The glycogen was first extracted from chicken liver. The extract was then divided into portions, one for each of the following: acid hydrolysis, enzymatic hydrolysis, Molisch¶s test and Iodine test. The qualitative test was composed of the Benedict¶s, Barfoed¶s, Seliwanoff¶s, Bial¶sOrcinol, Mucic Acid and Phenylhydrazone Test.
Carbohydrates are the most abundant organic compounds in the plant world. They act as storehouses (glucose, starch, glycogen) of chemical energy. Water-soluble carbohydrates often have a sweet taste and therefore are called sugars. Another term for carbohydrate is saccharide. The objective of this experiment are as follows: to isolate polysaccharides from animal and plant sources and explain the principle involved, to perform the general tests for carbohydrates, to compare the products of acid and enzymatic hydrolyses, to illustrate the specificity of alpha-amylase on the hydrolysis of the isolated polysaccharide, to prepare the dialyzing bag used in separating the products of enzymatic hydrolysis and to examine microscopically the different osazone and mucic acid crystals. Carbohydrates are actually polyhydroxyaldehydes and polyhydroxyketones, often but not always with the general formula (CH2O)n, where n equals 3 or more. Carbohydrates are divided into three general classes (Monosaccharide, Oligosaccharide, Polysaccharise) depending on the number of carbohydrate molecules they contain. Monosaccharides or simple sugars are those carbohydrates, which cannot be hydrolyzed into simpler compounds. It has a general formula of CnH2nOn, with one of the carbons being the carbonyl group of either an aldehyde (aldoses) or a ketone(ketoses). Aldehydes and ketones react with alcohols to form hemiacetals. Cyclic hemiacetals form very readily when hydroxyl and carbonyl groups are part of the same molecule and that their interaction produces a
ring.Glucose, fructose and xylose are common examples of monosaccharides. A common way of representing the cyclic structure of monosaccharides is the Haworth projection, named after the English chemist Sir Walter N. Haworth. In a Haworth projection, the ring is drawn flat and viewed through its edge, with the anomeric carbon on the right and the oxygen atom to the rear. A reducing sugar is any carbohydrate that reacts with a mild oxidizing agent under basic conditions to form an aldonic acid.(reduces the oxidizing agent). All of the examples already mentioned (glucose, fructose and xylose) are reducing sugars. Oligosaccharides contain 2-10 monosaccharide units. Among the oligosaccharides, sucrose and lactose are of considerable biological importance. Polysaccharides consist of large numbers of monosaccharide units bonded together by glycosidic bonds. Some examples of important polysaccharides are starch and glycogen. Glycogen acts as the energy-reserve carbohydrate for animals. It is particularly prevalent in liver and skeletal muscle. Liver glycogen is used to maintain a constant level of blood glucose.Starch is used for energy storage in plants. Hydrated glycogen and starch are digested by the endosaccharidase alpha-amylase of saliva and pancreatic juice. Hydration of the glycogen occurs during heating and is essential for digestion. Aside from the three general classes, carbohydrates can also be categorized as simple
4-glycosidic bond to carbon 4 of Dglucopyranose. which means that it contains five carbon atoms and it includes an aldehyde functional group. Seliwanoff¶s. The first class is a 2-step analysis consisting of the use of dehydrating acids followed by the condensation reagents.or complex.2-glycosidic bond. Figure 4. Structure of Sucrose Galactose is a sugar contained in milk. Structure of Glycogen . ketose oligosaccharide. or grape sugar and is one of the main products of photosynthesis and starts cellular respiration. and Iodine reaction test and standard carbohydrate solutions were subjected to qualitative tests which includes Benedict¶s. Lactose is the sugar found in mammalian milk. Lactose is a reducing sugar. The second class of test reagents uses solutions containing copper (II) ions to detect reducing sugars. Figure 3. the sample solution was subjected to the general test for polysaccharides. The result is a fanlike structure with one terminal reducing end and many non-reducing ends. aldose oligosachharide. Galactose makes up half of the sugar called lactose that is found in milk. Bial¶sOrcinol and Seliwanoff¶s Tests. Compounds tested (Samples used) Glucose is also known as D-glucose. Structure of Xylose Figure 7. The carbohydrate reduces the copper (II) ions into copper (I) oxide. It is a non-reducing sugar. dextrose. Figure 5. Complex carbohydrates are also known as starches and are made of three or more linked sugars. Structure of Lactose Sucrose is obtained prinicipally from the juice of sugar cane and sugar beets. Simple carbohydrates are also called simple sugars and are chemically made of one or two sugars. Structure of Glucose Fructose or fruit sugar is a simple ketose monosaccharide found in many foods. Bial¶s-Orcinol. which consists Molisch¶s. Since these aldehydes will condense with aromatic amines and phenols to give intensely colored compounds. Figure 2. EXPERIMENTAL A.6 linkages at the branch points. Barfoed¶s. Carbohydrates in the presence of non-oxidizing acids. Figure 6. Mucic Acid and Phenlhydrazone test. It is an aldopentose. In sucrose. Carbohydrate test reagents can be divided into general classes based on the type of reaction involved. carbon 1 of alpha-D-glucopyranose bonds to carbon 2 of D-fructofuranose by an alpha-1. In the experiment. fufural formation can be used a test for carbohydrates. It consists of D-galactopyranose bonded by a Beta1. This class includes the Benedict¶s and Barfoed¶s test.4 linkages and with alpha-1. Structure of Fructose Xylose is a sugar isolated from wood. undergo dehydration to form furfural or hydroxymethyl furfural. Structure of Galactose Glycogen has a branched structure with linear chains of consecutive glucose residues joined by alpha-1. This is the principle involved in Molisch¶s . Figure 1.
xylose. Complete hydrolysis of both amylose and amylopectin yields only D-glucose. unbranched chains of as manyas 4000 D-glucose units joined by alpha1. The mixture was then transferred into a small beaker and was boiled for 2 minutes. 4.01M I2 was added into 1mL sample solution and the result was noted. It was then placed in a warm water bath and was stirred until it became clear. The presence of a reducing sugar was then tested using the Benedict¶s test. the first one is the Molisch¶s test and the other is the Iodine Reaction test. 5 drops of each carbohydrate solution was placed in separate test tubes and was mixed with 1mL each of the following reagents: Benedict¶s. fructose. 2. After 24hours. 1mL of 0. The solution was mixed well and was covered with cotton. Procedure 1.3mL of saliva.4-glycosidic bonds. glycogen was extracted from chicken liver.4. About 3 grams of chicken liver was minced and was mixed with 12mL boiling water. 2mL concentrated H2SO4 was carefully poured down the side of the tube to form a layer. Then the glycogen precipitation by ethanol was performed. The Phenylhydrazone reagent was prepared by mixing 2g of phenylhydrazine hydrochloride. For the Molisch¶s test. To perform the enzymatic hydrolysis. a few drops of Molisch¶s reagent (5% alphanaphthol in 95% ethanol) were added into 1mL glycogen solution. 3g CH3COONa. Extraction of Glycogen: For this experiment. The mixture was then filtered and divided intoportions for the general test for polysaccharides.000 D-glucose units joined by alpha-1.glycosidic bonds. 2 drops of carbohydrate solution (glucose. covered with a marble and was kept in a water bath for 30 minutes. To do this. 10mL of the isolated carbohydrate was mixed with 2. It was then allowed to stand at room temperature for 30 minutes. fructose. The result was then noted.1% acetic acid was added to improve the precipitation of proteins. sucrose and starch) were mixed with 4 drops of freshly prepared phenylhydrazine reagent. The crystals were then observed under the microscope. 3. sucrose and starch. Amylose is composed of continuous. For the Iodine reaction test a few drops of 0. The opening of the test tube was . Hydrolysis of Polysaccharides To perform the acidic hydrolysis. General Test for Polysaccharides There are two general tests for polysaccharides. xylose. It was then heated in a boiling water bath for 30 minutes. The Mucic Acid test required the mixture of 3 drops of the carbohydrate solution (galactose and lactose) and 3 drops of concentrated HNO3 on a glass slide. namely glucose. The test tubes were placed in a boiling water bath and were removed after one solution already gave a positive result. The hydrolysate was then subjected to Benedict¶s test. Then. The specimen was heat fixed and was cooled at room temperature. it was heated in a boiling water bath for 30 minutes with constant addition of water to avoid drying.lactose.Sample Characteristics Glucose Fructose Xylose Lactose Sucrose Starch Simple Simple Simple Complex Complex Complex Carbohydrates and their Aldose Ketose Aldose Aldose Ketose Aldose Monosaccharide Monosaccharide Monosaccharide Oligosaccharide Oligosaccharide Polysaccharide Reducing Reducing Reducing Reducing Nonreducing Nonreducing B. 5 drops of concentrated HCl was mixed to 5mL of the isolate in a test tube. Amylopectin contains chains of as many as 10. The tubes Figure 8. Structure of Starch Table 1. This step would allow the precipitation of proteins. The solution was then introduced into a dialyzing bag and it was suspended overnight in a small flask with 50mLdistilled water. lactose. the dialyzing bag was discarded and the solution inside the flask was concentrated using an open flame to the volume of 10mL. Barfoed¶s. acidic and enzymatic hydrolyses. Seliwanoff¶s and Bial¶s-Orcinol test. 5-10 drops of ethanol was addedto 1mL glycogen solution. In different test tubes. Qualitative test for Carbohydrates This test used six different carbohydrate solutions. and 10mL distilled water. The mixture was grinded thoroughly until lumps were no longer visible.Starch can be separated into two principal polysasccharides: amylose and amylopectin.
the visible result should not be a blue-black complex and in the case of this particular experiment we noted a red solution for the test. RESULTS AND DISCUSSION: Table 2. Since hydrolysis should have converted the glycogen to glucose. Results for Hydrolysis and Benedict¶s test The Benedicts test both for the acid and enzymatic hydrolysate gave a false result. These products condense with -naphthol to yield a purple condensation product.were cooled and the crystals were observed under the microscope. concentrated sulfuric acid was used as the dehydrating acid. producing the blue-black color. this test can be used to distinguish starches from other carbohydrates. for example. nor do they hydrolyze maltoe. carbohydrate dehydration product purple product Either acids or enzymes catalyze the hydrolysis of the glycosidic bonds. Table 4. Several enzyme catalyzed hydrolyses are more specific with respect to bonds cleaved. If the saccharide is a reducing sugar. result from the reaction of the sulfuric acid with pentoses and/or hexoses. The positive result for the Iodine Reaction test is a blue-black complexwhich is only seen in starches. The dehydration products of carbohydrates. Thus glycogen is initially split by alpha-amylase action into branched dextrin¶s of medium molecular weight. glycogen can be separated from sugars and other water-soluble compounds by the precipitation with alcohol. hydrolyze alpha-1. Simpler oligosaccharides and monosaccharides do not form this complex with iodine. and amylopectin. Iodine forms a large complex polysaccharide with the -amylose helix.4 bonds. random hydrolysis of internal alpha-1. a red precipitate. only small amounts if maltose is formed. The result for Molisch¶s test was a purple product. which indicates the presence of glucose. Sugar Benedict s Barfoed s Seliwanoff s Bial sOrcinol Glucose Brick red Blue Orange Moss precipitate solution* solution green solution Fructose Brick Red Brick red Cherry red Dark precipitate precipitate solution brown solution Xylose Brick Red Brick red Green Blue precipitate precipitate solution green solution Lactose Brick Red Blue Orange Brownish . Results Carbohydrates for Qualitative Tests for Results with asterisk show false or wrong results. They do not however. it will reduce the copper(II) ions to copper(I) oxide. In the acid-catalyzed hydrolysis there is a random cleavage of bonds. Purified glycogen is obtained from aqueous solution by subsequent reprecipitation with alcohol. Benedict¶s test is used as a general test for detecting reducing sugars. so the test is used to distinguish between carbohydrates and noncarbohydrates. the Benedicts test should have given a brick-red precipitate. This acid dehydrates all carbohydrates. with the intermediate formation of all the various possible oligosaccharides and the final conversion of these oligosaccharides to glucose. which is a positive result for carbohydrates. Since the sample we used is not starch. Thus. regardless of molecular size. In the Molisch test. The extract was obtained based from the principle that polysaccharides are considerably less soluble than sugars in aqueous alcohol.6 linkages. Results for Extraction of Glycogen and General Tests for Polysaccharides Chicken Liver Description Yellow clear solution Molisch s Purple ring b/w H2SO4 and glycogen Iodine Red solution Results with asterisk show false or wrong results. The final degradation products of the action of alpha-amylase on glycogen are glucose. Description Benedicts Acid Chicken Liver: Green precipitate* yellow clear solution Enzymatic Starch: Blue Solution The table above shows the results obtained for the general test for polysaccharides. maltose and isomaltose. The alpha-amylase catalyze the rapid. a helical saccharide polymer. a reducing sugar. alpha-amylase of human saliva. furfural or 5hydroxymethylfurfural. R ± CHO reducing carbohydrates +2Cu2+ +5OH± R ± C O ±2 carbohydrate ion +Cu2O (brick red ppt)+ 3H2O In the Iodine reaction test starches form deeply colored blue-black complexes with iodine. Table 3. Starches contain -amylose. The glucose is formed by the relatively slow end cleavages of the oligosaccharides.
six carbohydrate solutions were subjected to different qualitative tests. The Barfoed¶s test employs the same reagent as the Benedict¶s test. Since lactose is a dimer of glucose and galactose. Lactose* For this part of the experiment. the rest were aldoses. Glucose as we know is also a reducing monosaccharide. only fructose and sucrose were ketoses. The osazones from different reducing sugars have characteristic crystalline forms and a definite formation time. copper (II) oxide. The Phenylhydrazone test can differentiate reducing sugars by the phenylhydrazones (osazones) they form with a phenylhydrazine reagent. This test can be used to distinguish between reducing monosaccharides and reducing disaccharides by observing the time that the red precipitate will form. Mucic Acid. Bial¶s test is used to distinguish between pentoses and hexoses. which indicates that they are reducing monosaccharides. Seliwanoff¶s test is used to distinguish between aldoses and ketoses. For the Barfoed¶s test. pentose dehydration product: condensation product furfural blue or green Galactose* Aldohexoses are converted to their corresponding dicarboxylic acids in the presence of strong oxidizing agents like concentrated HNO3. the brick red precipitate was produced within 3 minutes by fructose and xylose. The difference is that this ion is used in a slightly acidic medium. Reducing disaccharides cause the formation of copper(I) oxide after approximately 10 minutes. gave correct results. the discussion proceeds to Barfoed¶s test. while hexoses yield a muddy brown-to-gray condensation product. Seliwanoff¶s . Seliwanoff¶s test. The . Table 5. Reducing monosaccharides cause the formation of copper (I) oxide within 2± 3 minutes. The test to distinguish aldoses and ketoses. As discussed earlier the Benedict¶s test is used to determine reducing sugars by the appearance of a brick-red precipitate. The dicarboxylic acid (mucic acid) produced from the oxidation of galactose is relatively insoluble and separates out as colorless crystals. The sucrose and starch solution remained blue. When mixed with Seliwanoff¶s reagent. so only fructose and sucrose gave a cherry-red product. which was expected since they are not reducing sugars. The Mucic Results for this test should have shown white crystals both for lactose and galactose.Bial¶s-Orcinol. a reducing disaccharide should have also produced a brick-red precipitate within 10 minutes time. Pentoses subjected to the test yield a blue or green condensation product. and Phenylhydrazine test. it should have produced the same result as fructose and xylose. The carbohydrate solutions used all gave a correct result. Lactose.precipitate Sucrose Blue green solution Blue green solution solution* Blue solution Blue solution solution Cherry red solution Yellow solution Starch black solution Moss green solution Brown solution Acid test is based on the formation of a crystalline saccharic acid that is insoluble in dilute HNO3-a reaction that is unique to galactose and galactose containing compounds. ketopentoses and ketohexoses react within 2 minutes to form a cherry-red condensation product. The tests include the Benedict¶s. R ± CHO reducing saccharide + 2Cu + 2H2O R COOH + carboxylic +Cu2O (brick red ppt )+4H ± Seliwanoff¶s test uses 6M hydrochloric acid as the dehydrating acid and resorcinol as the condensation reagent. Among the samples used. ketose dehydration product cherry-red product(within 2 min) Bial¶s test uses concentrated hydrochloric acid as the dehydrating acid and orcinol with a trace of iron(III) chloride as the condensation reagent. Barfoed¶s. Results for Mucic Acid Test Results with asterisk show false or wrong results. but it also failed to do so in this experiment. Since the Benedict¶s test was already mentioned.
newagepublishers. Devlin. pp. W. Function & Informatics. Loffredo W.cerlabs.20-27 Xylose No picture was available Lactose Bettelheim.11 . Brooks/Cole (2010). Textbook of Biochemistry with clinical correlation 6thedition. Brown. Biomicromolecules: Introduction to Structure..Hoboken. Campbell. Organic & Biochemistry. pp. 1 -28 Clark J.pdf 2-18-11 Sucrose http://www. Farrel.O.com/samplechapte r/000091. (2007).Hoboken New Jersey.B.. pp. pp. Introduction to General. Results for Phenylhydrazone Test Glucose REFERENCES Fructose Books: C. Experimental Biochemistry.M. Wiley-Liss (2006).nitric acid will also hydrolyze the dimer and so lactose should have also given a positive result. Starch Table 6..Stan Tsai.154165 Cantarow A. pp. http://www. Biochemistry 3rd Edition.. New Jersey John Wiley & Sons Inc. 517 -539 Thomas M.1056-1127 Internet: Schreck J. Saunders Company (1962).pdf 2-19.com/experiments/108754044 64.
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