KWAME NKRUMAH UNIVERSITY OF SCIENCE AND TECHNOLOGY COLLEGE OF SCIENCE DEPARTMENT OF BIOCHEMISTRY INTRODUCTORY LABORATORY TECHNIQUES NAME: DAAYI BONIFACE INDEX NUMBER: 4057820 DATE: 10TH FEBRUARY 2021 TITLE: QUALITATIVE TESTS ON CARBOHYDRATES AIMS: • • • • • To test for carbohydrates using Molish test To test for monosaccharide using barfoed’s test To test for reducing sugars using benedict’s test To distinguish fructose from glucose using seliwanoff’s test To test for pentose using bial’s test INTRODUCTION: Carbohydrates are neutral compounds composed of carbon, hydrogen, and oxygen in which the H:O ratio is 2:1 as shown by the carbohydrates formula C n (H2O) n. Chemically they are polyhydroxy alcohols with potentially active aldehyde or ketone groups. Analytically, they are all reducing sugars or give rise to reducing sugars on hydrolysis. Biologically, they represent one of the three principal forms in which carbon is transported, stored and utilised in living things Fearon WF (1949). Introductory to Biochemistry (2nd edition). London: Heinemann, P.45. The carbohydrates are divided into four chemical groups, monosaccharaides, disaccharides, oligosaccharides and polysaccharides. Polysaccharides serve for the storage of energy and as structural components. Structural polysaccharides are frequently found in combination with proteins or lipids. The 5-crbon monosaccharaide ribose is an important component of coenzymes and the backbone of the genetic molecule known as RNA Fearon WF (1949). Introductory to Biochemistry (2nd edition). London: Heinemann, P.47 There are three types of carbohydrates namely: Monosaccharaide, oligosaccharide, and polysaccharides. Monosaccharaides are the simplest group of carbohydrates and often called simple sugars since they cannot be further hydrolysed. They possess a free aldehyde or ketone group and are colourless, crystalline solid which are soluble in water and insoluble in a non-polar solvent and examples include Glucose, Fructose, and Erythrulose Lehninger, A. L., Nelson, D. L., and Cox, M. M. (2000). Lehninger principles of biochemistry. New York: Worth publishers. Oligosaccharides are compound sugars that yield 2 to 10 molecules of the same or different monosaccharaides on hydrolysis. Based on the number of monosaccharaides units, it is further classified as disaccharide and examples include sucrose, lactose, and maltose Lehninger, A. L., Nelson, D. L., and Cox, M. M. (2000). Lehninger principles of biochemistry. New York: Worth publishers. Polysaccharides contain more than 10 monosaccharide units and can be hundreds of sugar units in length. They are primarily concerned with two important functions that is structural functions and the storage of energy and examples include starch, glycogen, cellulose and pectin Rodwell, V. W., Botham, K. M., Kennelly, P. J., Weil, P. A., and Bender, D. A. (2015). Harpers illustrated biochemistry (30th edition). New York. • THE MOLISH TEST This is the general test for all carbohydrates. Conc. H 2SO4 hydrates glycosidic bonds to yield monosaccharaides which in the presence of an acid get dehydrated to form furfural and its derivatives. These products react with sulphonated a-naphtol to give a purple complex Sadasivam, S. and Theymoli Balasubramanian (1985). Practical manual (undergraduate), TamilNadu Agricultural University, Coimbatore, p.2 MATERIALS: The materials used were, two sets of seven Test tubes labelled from A-G, Conc. H2SO4, anaphtol, pipettes. METHOD: 2ml of test solution was gently pipetted into separate test tubes labelled A-G. 2 drops of the Molish reagent was added. The solution was mix thoroughly and 1-2ml of conc. H2SO4 was poured slowly and carefully down the side to form a layer. The solution was observed carefully to see any change in colour at the junction two layers. Appearance of purple colour on the interface of the test solution shows that carbohydrate is present. RESULTS: The purple ring indicates the presence of carbohydrates in the solution. TEST 2ml sample A + 2 drops of Molish reagent + 1ml conc. H2SO4 OBSERVATION No colour change Formation of purple ring at interface + heat INFERENCE Carbohydrate present 2ml sample B + 2 drops of Molish reagent + 1ml conc. H2SO4 No colour change Formation of purple ring at interface+ heat Carbohydrate present 2ml sample C + 2 drops of Molish reagent + 1ml conc. H2SO4 2ml sample D + 2 drops of Molish reagent + 1ml conc. H2SO4 No colour change Formation of purple ring at interface+ heat No colour change Formation of purple ring at interface+ heat Carbohydrate present 2ml sample E + 2 drops of Molish reagent + 1ml conc. H2SO4 No colour change Formation of purple ring at interface+ heat Carbohydrate present 2ml sample F + 2 drops of Molish reagent + 1ml conc. H2SO4 No colour change Formation of purple ring at interface and then disappeared+ heat Carbohydrate present 2ml sample G + 2 drops of Molish reagent + 1ml conc. H2SO4 No colour change Carbohydrate absent Carbohydrate present DISCUSSION: All carbohydrates give a positive reaction for Molish test. It is based on the dehydration of the carbohydrates by sulphuric acid to produce an aldehyde, which condenses with two molecules of a-naphtol resulting in the appearance of a purple ring at interface Sadasivam, S. and Theymoli Balasubramanian (1985). Practical manual (undergraduate), TamilNadu Agricultural University, Coimbatore, p.5 Don’t add too much Molish reagent. Don’t pour sulphuric acid directly into the solution. Otherwise charring of carbohydrates will occur and a black ring will be formed, giving a false negative test Sadasivam, S. and Theymoli Balasubramanian (1985). Practical manual (undergraduate), TamilNadu Agricultural University, Coimbatore, p.3 There was no carbohydrate present in sample G because it was the test experiment. CONCLUSION: Formation of purple ring at the interface indicates the presence of carbohydrates. • BARFOED’S TEST This test is used for distinguishing monosaccharaides from reducing disaccharides. Monosaccharaides usually react in about 1-2 min while the reducing disaccharides take much longer time between 7-12 min to get hydrolysed and then react with the reagent. Brick red colour is obtained in this test which is due to the formation of cuprous oxide Vogel’s Textbook of Quantitative Chemical Analysis, 5th edition. MATERIALS: The materials used were, boiling water bath, barfoed’s reagent, test tubes, and pipette. METHOD: 1ml of sample solution was pipetted into the test tubes labelled A-G and 2ml of barfoed’s solution was also pipetted into the sample solution. The test tubes are then kept in a water bath. A briskly boiling water should be used for obtaining reliable results. Formation of brick red colour shows the presence of monosaccharaide. The test tubes should be in the water bath for about 15 mins. RESULTS: TEST 1ml sample A + 3ml of barfoed’s reagent + heat 1ml sample B + 3ml of barfoed’s reagent + heat 1ml sample C + 3ml of barfoed’s reagent + heat 1ml sample D + 3ml of barfoed’s reagent + heat 1ml sample E + 3ml of barfoed’s reagent + heat 1ml sample F + 3ml of barfoed’s reagent + heat 1ml sample G + 3ml of barfoed’s reagent + heat OBSERVATION Blue solution formed Red ppt formed at the bottom Blue solution formed Red ppt formed at the bottom Blue solution formed Red ppt formed at the bottom Blue solution formed Red ppt formed at the bottom Blue solution formed Red ppt formed at the bottom Blue solution formed Red ppt formed at the bottom Blue solution formed No red ppt formed INFERENCE Monosaccharaide present Monosaccharaide present Monosaccharaide present Monosaccharaide present Monosaccharaide present Monosaccharaide present Monosaccharaide absent DISCUSSION: Reddish brown precipitate is seen on the sides and bottom of the tube. The precipitate of the sides and bottom indicates the presence of monosaccharaides. Disaccharides are weak reducing agents and therefore do not reduce cupric ions in an acidic medium. Hence barfoed’s test is used to differentiate between disaccharides and monosaccharaides. Keep proper track of the boiling time. Allow gradual cooling at room temperature. If reheating is necessary, do it after the solution has become cold William H. Welker (1915). “A Disturbing factor in Barfoed’s test”. Monosaccharaide was not present in sample G because it was the test experiment. CONCLUSION: Formation of reddish brown ppt showed the present of monosaccharaide in the samples. • BIAL’S TEST This test is useful in the determination of pentose sugars. Reaction is due to formation of furfural in the acid medium which condenses with orcinol in presence of ferric ions to give a blue-green complex which is soluble in butyl alcohol Vogel’s Textbook of Quantitative Chemical Analysis, 5th edition. MATERIALS: The materials used were boiling water bath, Bial’s reagent, test tubes, and pipette and test solutions. METHOD: 3ml of bial’s reagent is added to the empty test tubes. 3ml of the test solution is then added to the above test tubes. The tests tubes are then heated in a water boiling bath. Allow the solution to cool at room temperature. It’s observed that a bluish colour appears in the test tube upon heating. RESULTS: The bluish colour indicates the presence of pentose sugar in the test solution TEST 2ml of sample A + 3ml of bial’s reagent + heat OBSERVATION Yellow colour is seen Brown colour change INFERENCE Hexose may be present 2ml of sample B+ 3ml of bial’s reagent + heat 2ml of sample C + 3ml of bial’s reagent + heat 2ml of sample D + 3ml of bial’s reagent + heat 2ml of sample E + 3ml of bial’s reagent + heat 2ml of sample F + 3ml of bial’s reagent + heat 2ml of sample G + 3ml of bial’s reagent + heat Yellow colour is seen No colour change Yellow colour is seen Brown colour change Yellow colour is seen No colour change Yellow colour is seen No colour change Yellow colour is seen Blue-green colour change Yellow colour is seen No colour change Pentose may be present Hexose may be present Hexose and pentose absent Hexose and pentose absent Pentose may be present Hexose and pentose may be absent DISCUSSION: The formation of a bluish product shows the positive test. All other colours indicate a negative result for pentose. And also hexoses react to form green, red, or brown products Baldwin, E. and Bell, D. J., Cole’s practical physiological chemistry, published by Heffer, Cambridge, (1955) p.189. In cooling the solution, it should not be rapid or else other results may be affected. CONCLUSION: The formation of a bluish product shows the positive result of a pentose present whiles any other colours show the presence of hexose. SELIWANOFF’S TEST This test is used to distinguish aldoses from ketoses. Ketoses undergo dehydration to give furfural derivatives which then condense with resorcinol to form a red complex. Prolonged heating will hydrolyse disaccharides and other monosaccharaides will also eventually give colour Vogel’s Textbook of Quantitative Chemical Analysis, 5th edition MATERIALS: The materials used were, Boiling water bath, Seliwanoff’s reagent, test tubes and pipette. METHOD: 1ml of each of the test solutions are pipetted into separate test tubes. Also a blank tube with 1ml of water is being prepared. 3ml of seliwanoff’s reagent is added to each tube and mixed and heated for exactly 30 seconds in a boiling water bath. The solutions are observed and time recorded. Heating is continued for 5 mins. Appearance of cherry red colour indicates fructose. RESULTS: The given solution contains a keto-sugar TEST 1ml of sample A + 3ml of seliwanoff’s reagent + 30 sec of heating +5 mins of heating 1ml of sample B + 3ml of seliwanoff’s reagent + 30 sec of heating +5 mins of heating 1ml of sample C + 3ml of seliwanoff’s reagent + 30 sec of heating +5 mins of heating 1ml of sample D + 3ml of seliwanoff’s reagent + 30 sec of heating +5 mins of heating 1ml of sample E + 3ml of seliwanoff’s reagent + 30 sec of heating +5 mins of heating 1ml of sample F + 3ml of seliwanoff’s reagent + 30 sec of heating +5 mins of heating 1ml of sample G + 3ml of seliwanoff’s reagent + 30 sec of heating +5 mins of heating DISCUSSION: OBSERVATION No colour change Colour changes to red Colour changes to dark brown No colour change No colour change Colour changes to cherry red No colour change Colour changes to cherry red Colour changes to dark brown No colour change No colour change Colour changes to cherry red No colour change No colour change Colour changes to cherry red No colour change No colour change Colour changes to dark green No colour change Changes to light brown Change colouration formed INFERENCE Ketose may be present Ketose or aldose may be present Ketose may be present Aldose or ketose may be present Aldose or ketose may be present Neither aldose nor ketose present Aldose may be present This test is used to distinguish between aldose and ketose sugars. When added to a solution containing ketoses, a red colour is formed rapidly indicating the positive test. Also during the test only aldose and ketose showed positive results from the formation of red colour solution. The test was done to show that ketoses are rapidly dehydrated and react faster than aldoses after seliwanoff’s reagent and the solutions in the test tube were heated in a water boiling bath Abramoff, Peter, Thomson, Robert (1966). An experimental approach to biology. WH Freeman and Company, San Francisco. P.47 Prolonged boiling will lead to the conversion of glucose to fructose resulting in a false positive test. Cooling must be gradual at room temperature Vogel’s Textbook of Quantitative Chemical Analysis, 5th edition. CONCLUSION: Formation of red colour product showed the presence of aldose and ketose. Aldose and ketose were not present in the sample G because it was the test experiment. REFERENCES: • • • • • • • • • Abramoff, Peter, Thomson, Robert (1966). An experimental approach to biology. WH Freeman and Company, San Francisco. P.47 Baldwin, E. and Bell, D. J., Cole’s practical physiological chemistry, published by Heffer, Cambridge, (1955) p.189. Fearon WF (1949). Introductory to Biochemistry (2nd edition). London: Heinemann, P.45) Fearon WF (1949). Introductory to Biochemistry (2nd edition). London: Heinemann, P.47 Lehninger, A. L., Nelson, D. L., and Cox, M. M. (2000). Lehninger principles of biochemistry. New York: Worth publishers. Rodwell, V. W., Botham, K. M., Kennelly, P. J., Weil, P. A., and Bender, D. A. (2015). Harpers illustrated biochemistry (30th edition). New York. Sadasivam, S. and Theymoli Balasubramanian (1985). Practical manual (undergraduate), TamilNadu Agricultural University, Coimbatore, p.2 Vogel’s Textbook of Quantitative Chemical Analysis, 5th edition. William H. Welker (1915). “A Disturbing factor in Barfoed’s test”.
