Qualitative Tests of Carbohydrates Experiment PDF

**Experiment No. 3** Qualitative Tests of Carbohydrates One of the most important constituents in our food is glucose which we usually obtain in the form of starch from plant sources. In our body glucose is readily utilized or is stored as glycogen. The metabolic processes in our body are mainly...

**Experiment No. 3** Qualitative Tests of Carbohydrates One of the most important constituents in our food is glucose which we usually obtain in the form of starch from plant sources. In our body glucose is readily utilized or is stored as glycogen. The metabolic processes in our body are mainly centred on glucose, which is a member of a large class of organic compounds called carbohydrates. These are generally referred to as sugars. Carbohydrates contain C, H and O atoms. Usually, H and O are present in the ratio of 2:1, just as in water; hence the name carbohydrates are in use. Carbohydrates in general have either an aldehyde group (as in glucose) or a keto group (as in fructose). Those containing an aldehyde group (as in glucose) are called as aldoses and those containing keto groups are called ketoses. They may also be referred to on the basis of the number of carbon atoms contained in them; for example, both glucose and fructose are hexoses as they have six carbon atoms in them. Glucose is the major source of energy whereas starch and glycogen function as storage polysaccharides in plants and animals respectively. The carbohydrates are also structural components of cell walls, connective tissues in animals and exoskeletons of invertebrates. **THEORY** Carbohydrates are optically active polyhydroxy aldehydes, polyhydroxy ketones or compounds, which give these units as hydrolysis product. Starch, cellulose and sugars are the familiar examples of carbohydrates. Carbohydrates are classified on the basis of number of polyhydroxy aldehyde or ketone units obtained from them on hydrolysis. Three broad classes are as follows: Monosaccharides are further classified on the basis of number of carbon atoms and functional group present in them. If a monosaccharide contains aldehydic group it is called aldose. If it contains keto group it is called ketose. Carbohydrates of all classes give Molisch's test. Carbohydrates, which are sweet in taste, are called sugars. Glucose, fructose (fruit sugar) and sucrose (table sugar) are examples of sugars. Sugars are classified into two major categories: reducing sugars and non-reducing sugars. Reducing property of sugars is detected by the three tests namely Fehling's test, Benedict's test and Tollen's test. Some of the different types of tests for detection of carbohydrates in the sample are as follows: **1) MOLISCH'S TEST** **    Principle** This is a general test for all carbohydrates. Alcoholic alpha naphthol forms furfural and furfural derivatives, such as hydroxymethylfurfural, by the concentrated sulphuric acid acting on the sugar. Conc. H~2~SO~4~ hydrates glycosidic bonds to yield monosaccharides which in the presence of an acid get dehydrated to form furfural and its derivatives. These products react with sulphonated α-naphthol to give a reddish-violet ring at the junction of the two liquids. Polysaccharides and glycoproteins also give a positive reaction. Molisch's reagent is 5% solution of alpha naphthol in alcohol. **    Reaction**                          **     ** **     Reagents** **Procedure and observations** Add 2-3 drops of α-naphthol solution to 2 ml of the test solution. Very gently pipette 1ml conc. H~2~SO~4 ~along the side if the test tube so that the two distinct layers are formed. Then erect the test tube slowly. Carefully observe any color change at the junction two layers. The formation of reddish violet ring at the junction of two liquids indicates the presence of carbohydrates. **Precautions** Concentrated solution of organic compounds may give a red instead of a violet colour due to the charring action of the sulphuric acid. In case of doubt the experiment should be repeated on a more diluted solution of the substance to be tested **2) ANTHRONE TEST** **Principle** Anthrone reaction is another general test for carbohydrates. In this the furfural produced reacts with anthrone to give bluish green colored complex. **Reaction**                      **        Materials and Reagents** 1. 2. 3. **Procedure and observations** Add 0.5 - 1 ml of the test solution to about 2 ml of anthrone reagent and mix thoroughly. Observe whether the color changes to bluish green. If not, examine the tubes again keeping them in boiling water bath for 10 min. **3) IODINE TEST** **Principle** The composition of the blue or red or wine red coloured substance is not well defined. This may be an adsorption complex of starch or dextrins or glycogen with iodine rather than a definite compound. Iodine reagent is 0.5 ml of iodine diluted to 5 ml with distilled water. Potassium iodide is added to the reagent solution in order to make the iodine more soluble in water. Iodine forms colored adsorption complexes with polysacchaides. Starch gives blue color with iodine, while glycogen reacts to form reddish brown complex. Hence it is useful, convenient and rapid test for detection of amylase, amylopectin and glycogen. **Reagents** 1. 2. **Procedure and observations** Add 1 or 2 drops of dilute iodine solution to 2-3 ml of dilute starch or dextrin or glycogen solution. A blue, red and brown colour develops in case of starch, dextrin and glycogen respectively. In case of starch, the blue colour disappears on heating and reappears on cooling. But the red colour and the brown colour in cases of dextrin and glycogen respectively, do not reappear on cooling as in case of starch. **4) Barfoed's Test** **Principle** This test is used for distinguishing monosaccharides from reducing disaccharides. Monosaccharides 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 color is obtained in this test which is due to the formation of cuprous oxide. **Reaction** (CH~3~COO)~2~Cu~2~ + H~2~O ↔ 2CH~3~COOH + Cu(OH)~2~ Cupric acetate Cupric hydroxide Cu(OH)~2~ ↔ CuO+H~2~O                          **        Materials and Reagents** 1. 2. **Procedure and observations** Mix 5 ml of Barfoed's reagent with 1 ml of carbohydrate solution in a test tube and heat in a boiling water bath for 10 min. Appearance of a red precipitate of cupric oxide (Cu20) indicates the presence of reducing sugar. Barfoed's reagent is prepared by dissolving 13.3 gm. of neutral copper acetate in 200 ml of water and then adding 1.8 ml of glacial acetic acid. Barfoed's test is also copper reduction test but this test differs basically from Fehling's test or Benedict's test as it is carried out in acidic medium instead of alkaline medium. Under acidic conditions the reduction takes place efficiently. Monosaccharide's respond quickly to the test whereas disaccharides respond slowly. When the sugar solution is boiled in contact with the reagent the disaccharide is hydrolysed by acetic acid present in the reagent and the positive test is obtained. Chloride interferes with this assay as it causes the formation of a green precipitate the urine cannot be tested by this method as it contains chloride. **5) SELIWANOFF'S TEST** **Principle** 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 hydrolyze disaccharides and other monosaccharides will also eventually give color. **                  Reaction    ** ** ** **Materials and Reagents** 1. 2. **Procedure and observations** To the sugar (2 ml) add 2 ml of seliwanoff s reagent. A blank without sugar should also be prepared to judge the colour change. Place the tubes in boiling water for exactly 1 min. Note the colour change, if any, and then continue the heating for 5 minutes and periodically observe the colour change. Seliwanoff s reagent is 0.5% resorcinol in conc. HCl diluted 1:1 with water. Ketoses (naturally fructose) give fiery red colour. Aldoses (glucose, etc.) give the test weakly and slowly. If the boiling is prolonged, positive test is obtained with glucose (or maltose) due to its partial conversion to fructose. This test is also given by sucrose which is hydrolysed during the course of the test yielding fructose as one of the products. **6) Fehling's Test** **Principle** Fehling's test is a specific and highly sensitive for detection of reducing sugars. Carbohydrates with free aldehyde or ketone groups reduce copper sulphate to cuprous oxide forming a yellow or brownish red coloured precipitate. Fehling's reagent is prepared freshly by mixing equal volumes of two stock solutions A and B. Solution A is 6.93 grams of CuSO4.5H2O per 100 ml of water and Solution B is 20 grams of KOH and 34.6 grams of sodium potassium tartarate (Rochelle salt) per 100 ml solution. Formation of yellow or red ppt of cuprous oxide denotes the presence of reducing sugars. Rochelle salt acts as the chelating agent in this reaction. **Reaction**                                      **Materials and Reagents** 1. 2. 3. 4. **Procedure and observations** Add a few drops of sugar solution at a time to 5 ml of Fehling's solution and heat the mixture after each addition. Mix thoroughly and place the test tubes in vigorously boiling water bath. The production of yellow or brownish red cuprous oxide precipitate indicates the presence of reducing sugar. **7) Benedict's test** **Principle** Benedict's test is more convenient and this reagent in more stable. In this method sodium citrate functions as a chelating agent. Presence of reducing sugars results in the formation of red ppt of cuprous oxide. **Reaction**                      **Materials and Reagents** 1. 2. 3. 4. **Procedure and observations** Add 5mL of Bennedict's qualitative reagent to the sugar solution, and place the test tube boiling water bath for 2 minutes. In case of reducing sugars there will be an appearance of red precipitate. Bennedict's qualitative solution is prepared by dissolving 173 gm. of sodium citrate and 100 gm. sodium carbonate and 100 ml of water, by heating. If there is any turbidity, it should be removed by filtration. Copper sulphate solution (17.3 gm. copper sulphate in 100 ml water) is slowly added with constant stirring to the citrate-carbonate solution and the volume is made up to 11. This test is based on the modification of Fehling's test by Benedict. The difficulties faced by Fehling's test are, therefore, not faced in case of Benedict's test. In the presence of even small quantities of reducing sugars the entire body of the solution will be filled with a precipitate which is red. In the case of non-reducing sugar (say sucrose) the solution will remain perfectly clean. This reagent is routinely used and found to be reliable in the examination of urine for pathological amounts of sugars. Observe for the formation of red precipitates whose appearance would suggest the presence of reducing sugars in the given or sample extract. **8) Picric acid test** **Principle** It is another test for detection of reducing sugars. The reducing sugars react with picric acid to form a red colored picramic acid. **Reaction**                                  **Materials and Reagents** 1. 2. 3. **Procedure and observations** Add 1 ml saturated picric acid to 1 ml of sample solution followed by 0.5 ml 10% Na~2~CO~3~. Heat the test tubes in a boiling water bath. Appearance of red color would indicate the presence of reducing sugars in the sample solution. **9) Bial's test for Pentoses** **Principle** 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 colored complex which is soluble in butyl alcohol. **Reaction**                          **        Materials and Reagents** 1. 2. **Procedure and observations** To 1 ml of sugar solution in a test tube add 3 ml of concentrated HC1 and 0.5 ml of Bial's reagent. Heat the tube in a boiling water bath for one minute. Record your observations with different sugars. The Bial's reagent is prepared by dissolving 3 gm of orcinol and 0.1 gm of ferric chloride in 100 ml of ethanol. This is a sensitive test for the detection of pentoses. Heating with strong acid converts the pentose to furfural which then reacts with the coloured compound produced when orcinol and ferric chloride react with each other. A blue green compound is finally formed. This reagent reacts with many sugars but under the condition described above only pentoses yield blue- green colour. **10) Tollen's Test** Tollen's reagent is ammoniacal solution of silver nitrate. A reducing sugar, reduces silver ion to metallic silver which gets deposited on the inner surface of the test tube in the form of silver mirror. The reaction occurs as follows:  **Procedure and observations** **Prepare Tollen's reagent by adding sodium hydroxide solution dropwise to 1 mL aqueous silver nitrate solution to get the precipitate of silver oxide. Now add ammonium hydroxide solution while shaking the mixture so that initially formed silver oxide precipitate dissolves. Add the reagent to the sugar solution contained in a test tube and warm the reaction mixture on a water bath. Formation of silver mirror on the walls of the test tube shows the presence of reducing sugar.** **Caution! Never heat the test tube on direct flame as it may cause explosion.** **11) Reduction Tests** Carbohydrates with free aldehyde or ketone groups have the ability to reduce solutions of various metallic ions. Confirmatory Tests: I. Phenyl hydrazine test: Take about 300 mg of phenyl hydrazine mixture (discussed below), to it add a few drops of glacial acetic acid and then 5 ml of sugar solution. Shake well and heat in a boiling water bath for 30 to 45 minutes. Take the tube out of the water bath and allow it to cool slowly. Yellow crystals of osazones will appear. Examine the crystals under the microscope and describe the nature of crystals. Phenyl hydrazine mixture is prepared by mixing equal weights of phenyl hydrazine hydrochloride and anhydrous sodium acetate. The mixing is to be done thoroughly in a mortar. Glucose and Fructose give identical osazones called glucosazones and fructosazones because excepting the first two carbons (which are used in formation of osazone) the remaining four carbon atoms have same configuration in both of them. Sucrose as such does not form any osazone because it has no reducing group available for reaction with phenyl hydrazine. On hydrolysis, however, it gives rise to osazone. Osazones of disaccharides are soluble in hot water. Therefore, in their cases the osazones do not precipitate during heating. They appear only after cooling.