Answer to Question #257583 in Biochemistry for Jai

Question

Perform the qualitative analysis of unknown carbohydrates using the biochemical tests and identify the nature of the unknown carbohydrate.

Answer 1

Qualitative Tests for Carbohydrates

Name of the test Application

1.     Molisch’s Test General test for carbohydrates

2.     Anthone Test General test for carbohydrates

3.     Iodine Test For glycan (starch, glycogen)

4.     Barfoed’s Test To distinguish between mono-saccharides from reducing disaccharides

5.     Seliwanoff’s Test For Ketones

6.     Fehling’s Test For reducing sugars

7.     Bendict’s Test For reducing sugars

8.     Picric acid Test For reducing sugars

9.     Bial’s Test For pentose

1) MOLISCH’S TEST

   Principle

This is a general test for all carbohydrates. Conc. H₂SO₄ 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-naphtha to give a purple complex. Polysaccharides and glycoproteins also give a positive reaction.

   Reaction

                       

 Reagents

1. Conc. H₂SO₄

2. α-naphthol: 5% (w/v) in ethanol (prepare fresh)

Procedure and observations

Add 2-3 drops of α-naphthol solution to 2 ml of the test solution. Very gently pipette 1ml conc. H₂SO₄ along the side if the test tube so that the two distinct layers are formed. Carefully observe any color change at the junction two layers. Appearance of purpose color indicates the presence of carbohydrates in the sample preparation or the test solution.

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.     Boiling water bath.

2.     Conc. H₂SO₄

3.     0.2% (w/v) anthrone solution

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

Iodine forms colored adsorption complexes with polysaccharides. 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.     Iodine solution: Prepare 0.005N iodine solution in 3% (w/v) potassium iodine solution.

2.     1% Test solutions of glucose, sucrose, starch, glycogen, cellulose etc.

Procedure and observations

Take 1 ml of the sample extract or test solution in a test tube. Add 4 - 5 drops of iodine solution to it and mix the contents gently. Observe if any colored product is formed. Note the color of the product.

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 hydrolyzed and then react with the reagent. Brick red color is obtained in this test which is due to the formation of cuprous oxide.

Reaction

(CH₃COO)₂Cu₂ + H₂O ® 2CH₃COOH + Cu(OH)₂

Cupric acetate Cupric hydroxide

Cu(OH)₂ ® CuO+H₂O

                        

       Materials and Reagents

1.     Boiling water bath

2.     Barfoed’s reagents: Dissolve 13.3 g of copper acetate in 200 ml water and add 1.8 ml of glacial acetic acid to it.

Procedure and observations

Take 2 ml of Barfoed’s solution in a test tube and add 1ml of sample solution to it. Keep the test tubes in a boiling water bath. A briskly boiling water bath should be used for obtaining reliable results. Look for the formation of brick red color and also note the time taken for its appearance.

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.     Boiling water bath

2.     Seliwanoff’s reagent: 0.05% (w/v) resorcinol in 3 HCl

Procedure and observations

Add 1ml of the test solution to 2 ml of Seliwanoff’s reagent and warm in a boiling water bath for 1min. Note for the appearance of a deep red color. This would indicate that the sample solution contains a keto sugar.

6) Fehling’s Test

Principle

Fehling’s test is a specific and highly sensitive for detection of reducing sugars. 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.     Boiling water bath.

2.     Fehling’s solution A: Dissolve 35 g of CuSO₄.5H₂O in water and make the volume to 500 ml.

3.     Fehling’s solution B: Dissolve 120 g of KOH and 173 g Na-K tartrate (Rochelle salt) in water and make the volume to 500 ml.

4.     Fehling’s reagent: Mix equal volumes of Fehling’s solution A and B. These solutions must be mixed immediately prior to use.

Procedure and observations

Add 1 ml of Fehling’s reagent (Reagent No. 4) to 1 ml of aliquot of the test solution. Mix thoroughly and place the test tubes in vigorously boiling water bath. Look out for the formation of red ppt of cuprous oxide which would indicate the presence of reducing sugars in the solution.

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.     Boiling water bath.

2.     Benedict’s reagents: Dissolve 173 g of sodium citrate and 100 g of anhydrous Na₂CO₃ in 600 ml of hot H₂O. Dilute to 800 ml with water.

3.     Dissolve 17.3 g of CuSO₄.H₂O in 100 ml hot water. Cool and dilute to 100 ml.

4.     Add Reagent No.2 to Reagent No.3 slowly with constant stirring. Make the final volume to 1 L.

Procedure and observations

Add 0.5 - 1 ml of the test solution or sample extract to 2 ml of Benedict’s reagent (Reagent No. 4). Keep the test tubes in a vigorously boiling water bath. 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.     Boiling water bath.

2.     Saturated picric acid: Dissolve 13 g picric acid in distilled water, boil and cool.

3.     10% Na₂CO₃.

Procedure and observations

Add 1 ml saturated picric acid to 1 ml of sample solution followed by 0.5 ml 10% Na₂CO₃. 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

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.     Boiling water bath

2.     Dissolve 1.5 g of orcinol in 100 ml of conc. HCl and add 20-30 drops of 10 % ferric chloride solution to it.

Procedure and observations

To 2 ml of Bial’s reagent add 4-5 drops of test solution and heat in a boiling water bath. Observe for the formation of blue-green colored complex.