14.1: Detection of Carbohydrates Lab Procedure

Learning Objectives

• Distinguish between reducing and non-reducing sugars using the Benedict's test.

 

Background

All carbohydrates consist of carbon, hydrogen, and oxygen atoms and are polyhydroxy aldehydes or polyhydroxyl ketones are compounds that can be broken down to form them. Examples of carbohydrates include starch, fiber, the sweet-tasting compounds called sugars, and structural materials such as cellulose.

 

Classification of Monosaccharides

The simplest carbohydrates—those that cannot be hydrolyzed to produce even smaller carbohydrates—are called monosaccharides. Two or more monosaccharides can link together to form chains that contain from two to several hundred or thousand monosaccharide units. Prefixes are used to indicate the number of such units in the chains. The most important naturally occurring monosaccharides contain three to six carbon atoms per molecule Monosaccharides of specific sizes may be indicated by names composed of a stem denoting the number of carbon atoms and the suffix -ose. For example, the terms triose, tetrose, pentose, and hexose signify monosaccharides with, respectively, three, four, five, and six carbon atoms. Monosaccharides are also classified as aldoses or ketoses. Those monosaccharides that contain an aldehyde functional group are called aldoses; those containing a ketone functional group on the second carbon atom are ketoses.

Although a variety of monosaccharides are found in living organisms, three of these hexoses are particularly abundant: D-glucose (found in fruit juices, honey and corn syrup), D-galactose (released when lactose is hydrolyzed), and D-fructose (found in honey, sweet fruits).

D- and L-glyceraldehyde provide reference points for designating and drawing all other monosaccharides. Sugars whose Fischer projections terminate in the same configuration as Dglyceraldehyde are designated as D sugars; those sharing the same configuration as Lglyceraldehyde are designated as L sugars.

 

Haworth Structures

Glucose tends to exist in the cyclic form, which is depicted by Haworth structure. The structure shown on top with the OH group on the first carbon atom projected downward, represent what is called the alpha (α) form. The structures on the bottom, with the OH group on the first carbon atom pointed upward, is the beta (β) form.

 

Disaccharides

Disaccharides (C12H22O11) are sugars composed of two monosaccharide units that are joined by a carbon–oxygen-carbon bond known as a glycosidic bond. Examples of disaccharides are maltose, sucrose (table sugar) and lactose (milk sugar).

 

Polysaccharides

Polysaccharides are very large polymers composed of thousands of monosaccharides joined together by glycosidic bonds. The three most abundant polysaccharides are starch, glycogen, and cellulose.

Starch is the most important source of carbohydrates in the human diet and accounts for more than 50% of our carbohydrate intake. Starch is a mixture of two polymers: amylose and amylopectin. Natural starches consist of about 10%–30% amylase and 70%–90% amylopectin.

Amylose is a linear polysaccharide composed entirely of D-glucose units joined by the α-1,4- glycosidic bonds.

Amylopectin is a branched-chain polysaccharide composed of glucose units linked primarily by α-1,4-glycosidic bonds but with occasional α-1,6-glycosidic bonds, which are responsible for the branching.

 

Cellulose

Cellulose, a fibrous carbohydrate found in all plants, is the structural component of plant cell walls. Because the earth is covered with vegetation, cellulose is the most abundant of all carbohydrates, accounting for over 50% of all the carbon found in the vegetable kingdom. Cotton fibrils and filter paper are almost entirely cellulose (about 95%), wood is about 50% cellulose, and the dry weight of leaves is about 10%–20% cellulose.

 

Benedict’s Test for Reducing Sugars

The chemical behavior monosaccharides are determined by their functional groups. An important reaction of monosaccharides is the oxidation of the aldehyde group, one of the most easily oxidized organic functional groups. Aldehyde oxidation can be accomplished with any mild oxidizing agent, such as Tollens’ reagent or Benedict’s reagent. With Benedict’s reagent, complexed copper (II) ions are reduced to copper (I) ions that form a brick-red precipitate of copper (I) oxide.

Any carbohydrate capable of reducing either Tollens’ or Benedict’s reagents without first undergoing hydrolysis is said to be a reducing sugar.

Figure \(\PageIndex{1}\): Benedict’s test was performed on three carbohydrates, depicted from left to right: fructose, glucose, and sucrose. The solution containing sucrose remains blue because sucrose is a nonreducing sugar.

 

Experimental Procedure

Question

Are there simple reducing sugars in my juice?

Diabetes mellitus is a disease that refers to the inability of the cells to take in glucose. The word diabetes refers to urination and mellitus refers to sweetness. Since the cells of diabetics cannot remove glucose from the blood, there is an excess of glucose circulating that is eliminated in the urine. The traditional method of diagnosing someone with diabetes mellitus was to taste the sweetness of the patient’s urine. Let’s use Benedict’s test for the detection process instead of the unhygienic alternative.

Make a hypothesis and ask what we would predict from a Benedict’s test if testing a urine sample of someone with diabetes mellitus.

 

Materials and Equipment

Test tubes, test tube rack, 400-mL beaker, droppers, hot plate, 5- or 10-mL graduated cylinder, Benedict’s reagent, apple juice, 2% carbohydrate solutions: glucose, fructose, sucrose, lactose and starch

 

Benedict’s Test for Reducing Sugars Procedure

1. Obtain 7 test tubes and number them 1-7.
2. Add to 10 drops of the materials to be tested (see Table 1) to each tube. Your instructor may ask you to test some additional materials. If so, include additional numbered test tubes.
3. Indicate in the table whether the sample you are testing is positive control, a negative control or experimental.
4. Before you begin the heating of the samples, use predict the color change (if any) for each sample. (use the sample type to aid in your prediction)
5. Add 40 drops (or 2 ml) Benedict’s solution to each tube.
6. Place all of the tubes in a boiling water bath for 3 min or until a noticeable color change and observe colors during this time.
7. After 3 min, remove the tubes from the water bath and let them cool to room temperature. Record the color of their contents in your data table.

 

Iodine Test for Starch Procedure

Carbohydrates that are used for energy storage are not reducing sugars since they are polymers that lack free aldehydes. Plant cells store energy in the form of starches like amylose and amylopectin. Since these molecules are larger than monosaccharides or disaccharides, they are not sweet to the taste and are not very soluble in water. An iodine/potassium iodine (KI I2) staining distinguishes starch from monosaccharides, disaccharides, and other polysaccharides. The basis for this test is that starch is a coiled polymer of glucose — iodine interacts with these coiled molecules and becomes bluish black. Iodine does not react with other carbohydrates that are not coiled, and remains yellowish brown. Therefore, a bluish black color is a positive test for starch, and a yellowish-brown color (i.e., no color change) is a negative test for starch. Notably, glycogen, a common energy storage polysaccharide in animals, has a slightly different structure than does starch and produces only an intermediate color reaction.

1. Obtain 7 test tubes and number them 1-7.
2. Hypothesis Testing: Indicate in the table if the sample is experimental or control. Predict your expected color changes for each sample.
3. Add to each tube the materials to be tested as indicated in the Table 2 below.
4. Your instructor may ask you to test some additional materials. If so, include additional numbered test tubes.
5. Add 10 drops of iodine to each tube. This test does NOT require boiling. Record the color of the tubes’ contents in the table below.