Qualitative Tests

Several qualitative tests have been devised to detect members of this biologically significant class of compounds. These tests will utilize a test reagent that will yield a color change after reacting with specific functional groups of the compounds being tested. The following exercises are reactions that can detect the presence or absence of carbohydrates in test solutions. They range in specificity to the very general (i.e., Molisch test for carbohydrates) to the very specific (i.e., mucic acid test for galactose).

Exercise: You are given solutions containing: fructose, glucose, lactose, galactose, ribose, ribulose, sucrose, and starch. Devise a scheme by which you can systematically identify these compounds.

Procedure
Perform the following qualitative tests on 0.2 M solutions (unless otherwise stated) of starch, sucrose, glucose, lactose, galactose, ribose, and ribulose. Use the scheme you devised in the prelab section to identify an unknown solution. The unknown will be 1 of the above solutions or a mixture of 2 of the above solutions.
Test 1. Molisch Test for Carbohydrates
The Molisch test is a general test for the presence of carbohydrates. Molisch reagent is a solution of alpha-naphthol in 95% ethanol. This test is useful for identifying any compound that can be dehydrated to furfural or hydroxymethylfurfural in the presence of H₂SO₄. Furfural is derived from the dehydration of pentoses and pentosans, while hydroxymethylfurfural is produced from hexoses and hexosans. Oligosaccharides and polysaccharides are hydrolyzed to yield their repeating monomers by the acid. The alpha-naphthol reacts with the cyclic aldehydes to form purple condensation products. Although this test will detect compounds other than carbohydrates (i.e., glycoproteins), a negative result indicates the absence of carbohydrates.

Method: Add 2 drops of Molisch reagent to 2 mL of the sugar solution and mix thoroughly. Incline the tube, and gently pour 5 mL of concentrated H₂SO₄ down the side of the test tube. A purple color at the interface of the sugar and acid indicates a positive test. Disregard a green color if it appears.

Test 2. Benedict’s Test for Reducing Sugars
Alkaline solutions of copper are reduced by sugars that have a free aldehyde or ketone group, with the formation of colored cuprous oxide. Benedict’s solution is composed of copper sulfate, sodium carbonate, and sodium citrate (pH 10.5).

The citrate will form soluble complex ions with Cu++, preventing the precipitation of CuCO₃ in alkaline solutions.

Method: Add 1 mL of the solution to be tested to 5 mL of Benedict’s solution, and shake each tube. Place the tube in a boiling water bath and heat for 3 minutes. Remove the tubes from the heat and allow them to cool. Formation of a green, red, or yellow precipitate is a positive test for reducing sugars.

Test 3. Barfoed’s Test for Monosaccharides
This reaction will detect reducing monosaccharides in the presence of disaccharides. This reagent uses copper ions to detect reducing sugars in an acidic solution. Barfoed’s reagent is copper acetate in dilute acetic acid (pH 4.6). Look for the same color changes as in Benedict’s test.

Method: Add 1 mL of the solution to be tested to 3 mL of freshly prepared Barfoed’s reagent. Place test tubes into a boiling water bath and heat for 3 minutes. Remove the tubes from the bath and allow to cool. Formation of a green, red, or yellow precipitate is a positive test for reducing monosaccharides. Do not heat the tubes longer than 3 minutes, as a positive test can be obtained with disaccharides if they are heated long enough.

Test 4. Lasker and Enkelwitz Test for Ketoses The Lasker and Enkelwitz test utilizes Benedict’s solution, although the reaction is carried out at a much lower temperature. The color changes that are seen during this test are the same as with Benedict’s solution. Use dilute sugar solutions with this test (0.02 M).

Method: Add 1 mL of the solution to be tested to 5 mL of Benedict’s solution to a test tube and mix well. The test tube is heated in a 55°C water bath for 10–20 minutes. Ketopentoses demonstrate a positive reaction within 10 minutes, while ketohexoses take about 20 minutes to react. Aldoses do not react positively with this test.

Test 5. Bial’s Test for Pentoses
Bial’s reagent uses orcinol, HCl, and FeCl3. Orcinol forms colored condensation products with furfural generated by the dehydration of pentoses and pentosans. It is necessary to use dilute sugar solutions with this test (0.02 M).

Method: Add 2 mL of the solution to be tested to 5 mL of Bial’s reagent. Gently heat the tube to boiling. Allow the tube to cool. Formation of a green solution or precipitate denotes a positive reaction.

Test 6. Mucic Acid Test for Galactose
Oxidation of most monosaccharides by nitric acid yields soluble dicarboxylic acids. However, oxidation of galactose yields an insoluble mucic acid. Lactose will also yield a mucic acid, due to hydrolysis of the glycosidic linkage between its glucose and galactose subunits.

Method: Add 1 mL of concentrated nitric acid to 5 mL of the solution to be tested and mix well. Heat on a boiling water bath until the volume of the solution is reduced to about 1 mL. Remove the mixture from the water bath and let it cool at room temperature overnight. The presence of insoluble crystals in the bottom of the tube indicates the presence of mucic acid.

Test 7. Iodine Test for Starch and Glycogen
The use of Lugol’s iodine reagent is useful to distinguish starch and glycogen from other polysaccharides. Lugol’s iodine yields a blue-black color in the presence of starch. Glycogen reacts with Lugol’s reagent to produce a brownblue color. Other polysaccharides and monosaccharides yield no color change; the test solution remains the characteristic brown-yellow of the reagent. It is thought that starch and glycogen form helical coils. Iodine atoms can then fit into the helices to form a starch-iodine or glycogen-iodine complex. Starch in the form of amylose and amylopectin has less branches than glycogen. This means that the helices of starch are longer than glycogen, therefore binding more iodine atoms. The result is that the color produced by a starch-iodine complex is more intense than that obtained with a glycogen-iodine complex.

Method: Add 2–3 drops of Lugol’s iodine solution to 5 mL of solution to be tested. Starch produces a blue-black color. A positive test for glycogen is a brown-blue color. A negative test is the brown-yellow color of the test reagent.