Carbohydrates and Reducing Sugar Determination

Objective:

To test a variety of carbohydrates to determine whether or not they are reducing sugars. Carbohydrates are divided into three main groups, monosaccharides, oligosaccharides and polysaccharides. Monosaccharides are simple sugars that cannot be hydrolyzed into simpler compounds (glucose, fructose, and galactose). Oligosaccharides yield two to ten simple sugars on hydrolysis (sucrose, maltose, lactose). Polysaccharides yield a large number of sugars upon hydrolysis (starch). Sugars that contain aldehyde groups that are oxidized to carboxylic acids are classified as reducing sugars. Reducing sugars participate in nonenzymatic, Maillard browning. Review the carbohydrate chemistry in your text book.

Fehlings test for reducing sugars

The reducing ability of sugars can be determined by their ability to reduce cupric ions in alkaline solution to form red cuprous oxide in the reaction: 2 Cu+2 + reducing sugar -------------- Cu2O (ppt) + oxidized sugar Blue solution Red precipitate

The best-known reagent for this test is Fehlings solution. It is made initially as two separate solutions, known as Fehling I and Fehling II. Fehling I is a blue aqueous solution of copper (II) sulfate, while Fehling II is a clear solution of aqueous potassium sodium tartrate and a strong NaOH. Equal volumes of the two mixture are mixed together to get the final blue Fehlings reagent. A positive reaction is shown by a green, yellowish orange, or red coloration of precipitate, depending upon the amount and kind of reducing sugars. The degree of color change from blue to red is an indication of the extent of the reduction and therefore a measure of the reducing power of the sugar. Besides being a general test for the reducing sugars, Fehlings can be used to screen for glucose in urine to detect diabetes. Another use is to determine dextrose equivalent (DE) of the starching sugar in the breakdown of starch to glucose syrup and maltodextrins in order to measure the amount of reducing sugar.

Safety: NaOH is very corrosive, causes severe burns and permanent eye damage.
Very harmful by ingestion and precautions should be taken as such not to come into direct contact. Copper(II) sulfate is also harmful if ingested.

Apparatus :

Test tubes/graduated cylinder Water bath, 37 C Water bath, boiling Mechanical pipettor Glucose/Fructose/sucrose/lactose/mannitol/starch, soluble HCl, 0.1N NaOH, 0.5N Starch solution (1%, dissolve in boiling water) Amylase solution (1% powdered amylase in cold water, 22 units/mg

Chemicals and Reagents:

solid) Fehlings solution I and II

Preparation of Fehlings reagent: Prepare fresh daily.

Fehling I: 7 g of hydrated copper (II) sulfate (CuSO4-5H2O) dissolved in 100 ml distilled water. Fehling II: 35 g of potassium sodium tartrate and 10 g of NaOH in 100 ml distilled water. Fehlings reagent: Equal volume of Fehling I and II are mixed to form a deep blue solution.

Procedure:
1. Transfer 3 ml starch solution to each of two test tubes. 2. Add 5 drops of amylase solution to one of the starch tubes from step 1 and incubate both tubes for 15 min in a 37 C water bath ( polysaccharide starch will be hydrolyzed by amylase to mono and oligosaccharides). 3. Prepare 0.1 M solutions of glucose, fructose, sucrose, lactose, mannitol. 4. Prepare 15 ml of 0.05 M sucrose-HCl solution by mixing 7.5 ml sucrose solution (0.1M) and 7.5 ml HCl (0.1N). 5. Transfer 5 ml aliquots of the sucrose-HCl solution (from step 4) to two test tubes. 6. Heat one of the test tubes from step 5 in a boiling water bath for 10 min; cool. Keep the other at room temperature. Neutralize the HCl in the tubes by adding 0.5 ml 0.5 N NaOH to each tube. 7. Transfer 3 ml of each solution into separate test tubes (step 3), including two starch solution tubes (step 1 and 2), and the heated and unheated sucrose solutions (step 5 and 6). Blank tube should be prepared with 3 ml distilled water. (Total 10 tubes) 8. Add 4 drops each of Fehling solutions I and II to each tube. Mix well. 9. Place the tubes in a boiling water bath for 3 min. 10.Remove the tubes from water bath and observe the color and appearance of the solution.

Questions:
1. 2. 3. 4. 5. 6. Draw the structure of each of the sugars you tested. Label them reducing or non reducing. What structure is characteristic of reducing sugars? Explain. Compare your results from the three sucrose solutions (in water, in HCl, in HCl with heating). Explain any difference. Fructose is not an aldose, yet it is considered a reducing sugar. Explain. List at least two disaccharides that are reducing sugars. Is starch reducing? Explain. Is starch treated with amylase reducing? Explain.

Suggested Reading:
1. BeMiller, J.N.; Whistler, R. L. Carbohydrates. In Food Chemistry, 3rd ed. 2. Fennema, O. R. Ed.; Marcel Dekker: New York, 1996; pp. 157-223.