Carbohydrates as Reducing Sugars

                                                                             Primary Concept                                                   

                                                A reducing sugar is any sugar that, in a solution, has an aldehyde or a ketone group. The enolization of sugars under alkaline conditions is an important consideration in reduction tests. The ability of a sugar to reduce alkaline test reagents depends on the availability of an aldehyde or keto group for reduction reactions. A number of sugars especially disaccharides or polysaccharides have glycosidic linkages which involve bonding a carbohydrate (sugar) molecule to another one, and hence there is no reducing group on the sugar; like in the case of sucrose, glycogen, starch and dextrin. In the case
of reducing sugars, the presence of alkali causes extensive enolization especially at high pH and temperature. This leads to a higher susceptibility to oxidation reactions than at neutral or acidic pH. These sugars, therefore, become potential agents capable of reducing Cu+2 to Cu+, Ag+ to Ag and so fort. Most commonly used tests for detection of reducing sugars are Fehling’s Test, Benedict’s Test and Barfoed’s Test.

1. Fehling’s Test
2. Barfoed’s Test
3. Seliwanoff’s Test
4. Bial’s Test

Fehling’s Test

 

Fehling’s Solution (deep blue colored) is used to determine the presence of reducing sugars and aldehydes. Perform this test with fructose, glucose, maltose and sucrose.

Procedure:

- To 1 mL of Fehling’s solution A (aqueous solution of CuSO4) add 1 mL of Fehling solution B (solution of potassium tartrate).
- Add 2 mL of the sugar solution, mix well and boil.

•  Try to see the red precipitate of cuprous oxide that forms at the end of the reaction.

Questions:

•  Write the reaction(s) involved in Fehling’s Test.
•  What is the function of tartrate?
•  Some disaccharides such as maltose are reducing agents, whereas others, such as sucrose are not. Explain briefly by incluiding the structures of the sugars.

Barfoed’s Test

Barfoed’s reagent, cupric acetate in acetic acid, is slightly acidic and is balanced so that is can only be reduced by monosaccharides but not less powerful reducing sugars. Disaccharides may also react with this reagent, but the reaction is much slower when compared to monosaccharides. Perform this test with glucose, maltose and sucrose.

Procedure:

- To 1-2 mL of Barfoed’s reagent, add an equal volume of sugar solution.
- Boil for 5 min. in a water bath and allow to stand.

• You will observe a brick-red cuprous oxide precipitate if reduction has taken place.

Questions:

• Write the reaction(s) involved in the Barfoed’s Test.
• When you test starch with Barfoed’s reagent, what would be the answer, positive or negative? Explain your answer by giving reasons and structures.

Seliwanoff’s Test

Seliwanoff’s Test distinguishes between aldose and ketose sugars. Ketoses are distinguished from aldoses via their ketone/aldehyde functionality. If the sugar contains a ketone group, it is a ketose and if it contains an aldehyde group, it is an aldose. This test is based on the fact that, when heated, ketoses are more rapidly dehydrated than aldoses. Perform this test with glucose, fructose, maltose and sucrose.

Procedure:

- Heat 1 mL of sugar solution with 3 mL Seliwanoff’s reagent (0.5 g resorcinol per liter 10% HCl) in boiling water.

• In less than 30 seconds, a red color must appear for ketoses.
• Upon prolonged heating, glucose will also give an appreciable color.

Questions:

• Write the reaction(s) involved in Seliwanoff’s Test.
• What is the funtion of resorcinol?
• What is the aim of using a strong acid?
• What is the result of testing sucrose with Seliwanoff’s reagent? Explain your answers by giving reasons and structures.

Bial’s Test

Bial’s Test is to determine the presence of pentoses (5C sugars). The components of this reagent are resorcinol, HCl, and ferric chloride. In this test, the pentose is dehydrated to form furfural and the solution turns bluish and a precipitate may form. Perform this test with ribose and glucose.

Procedure:

- To 5 mL of Bial’s reagent, add 2-3 drops of sugar solution and boil.

• Upon boiling, note the green-blue color formed.

Questions:

• Write the reaction(s) involved in Bial’s Test.
• Is it possible to distinguish DNA and RNA structures by using Bial’s Test?
• The boiling step is common for each test for the reducing sugars. Why boiling is necessary for the reduction to take place?