Lab 2: Reactions and Stereochemistry of Carbohydrates

Chemistry 103

Lab 2: Reactions and stereochemistry of carbohydrates

Objective: To determine the identities of several carbohydrates through a series of tests.

Introduction:

The Sugars:

Carbohydrates, also known as sugars or saccharides, are an important class of biochemical compounds, because they function as sources of energy in living organisms as well as structural materials for cells. Carbohydrates are complex biomolecules that contain both alcohol and carbonyl functional groups.

Polysaccharides can be viewed as polymers of various monosaccharide units. Monosaccharides are carbohydrates that cannot be broken down into smaller carbohydrate units. Two examples of monosaccharides, glucose (1) and fructose (2), are shown below.

Since glucose and fructose have both alcohol and carbonyl functionalities, they are able to form cyclichemiacetal and hemiketal compounds. The hemiacetal and hemiketal forms are in equilibrium with the open chain forms. Since both of these forms exist, one could predict that the chemistry of carbohydrates would be similar to the chemistry of ketones or aldehydes. In the case of glucose, its open chain form with the aldehyde should be easily oxidized. Carbohydrates that can be oxidized are known as reducing sugars. Carbohydrate chemistry is reviewed in Timberlake.

A disaccharide is a carbohydrate composed of two monosaccharide units. One such compound is sucrose (3), also known as table sugar. Sucrose is composed of a fructose unit and a glucose unit. The key point to note is that the fructose and glucose units are joined by an acetal link involving both the carbonyl carbons, which means that the aldehyde and ketone forms are unavailable to sucrose. Therefore, since sucrose is not in equilibrium with any aldehyde forms, sucrose is considered a non-reducing sugar.

In contrast, lactose (4) is a disaccharide composed of a galactose unit and a glucose unit. In lactose, the hemiacetal bond is formed with an alcohol oxygen of glucose and therefore the aldehyde functionality of the glucose unit is available. Thus, lactose is a reducing sugar.

Sucralose (Splendatm) is an artificial sweetener that has recently become popular. Its shape is similar to sucrose causing to bind to the sweetness-sensing receptors in our tongues. Although this compound somewhat mimics the flavor of sucrose, the structure is different enough that our bodies do not break it down therefore it provides us with no calories.

Sucralose

Polysaccharides, on the other hand, are used for energy storage. Amylose, shown below, is a type of starch. As you can see, starches generally contain long chains of glucose units.

The Tests:

In this experiment, you will test glucose, fructose, sucrose, sucralose and starch for reducing action by reacting them with Tollen’s and Benedict’s reagents. In the Tollen’s test, silver ion is reduced to metallic silver. The silver will stick to the side of the test tube giving it a mirror-like appearance. Be aware that the silver is very difficult to remove from the test tube generally ruining the tube. In the Benedict’s test, copper (II) is reduced to copper (I); there should be a definite color change. The results of the Tollen’s and Benedict’s tests should help you identify which unknowns are reducing sugars and which are non-reducing sugars.

The starch can be identified by its colorful interaction with iodine. When a couple of drops of iodine solution are added to a starch solution a dramatic dark blue color results. When the iodine solution is added to the glucose, fructose, sucrose and sucralose solutions, there should be little to no color change.

A few drops of iodine in a starch solution.

The body uses the monosaccharide glucose as its source of energy. In order for energy to be obtained from sucrose, starches and other saccharides, the organism must first break them down into glucose. This reaction entails the hydrolysis of the glycosidic linkages catalyzed by a collection of enzymes. Enzymes are proteins that accelerate biochemical reactions. These enzymes tend to be quite selective. For instance, lactase is used to break down lactose, amylase is used to break down amylose and invertase (also known as sucrase) is one of the enzymes that can hydrolyze sucrose to glucose and fructose. Benedict’s, Tollen’s and the iodine test should yield identical results for sucrose and sucralose however the invertase should hydrolyze sucrose while leaving sucralose intact. Following action of the enzyme upon sucrose, Benedict’s and Tollen’s tests should now yield positive results.

Glucose and fructose should yield identical results for all of the above listed tests. Polarimetry can be used to determine which is which. Fructose and glucose are chiral molecules (wikipedia.org has an excellent explanation of chirality) and therefore they have the ability to rotate plane-polarized light, a phenomenon that can be measured by a polarimeter. Glucose rotates plane-polarized light in a quite positive (+) direction (as viewed from detector to source) while fructose does so in a quite negative (–) direction allowing us to differentiate between the two.

Pre-Lab Questions and Activities:

Prepare your lab notebook with a table of the tests you will be running.

List the hazardous chemicals we will be working with.
What function does the invertase enzyme perform?
Which materials will be reducing sugars?

Materials

Glucose solution*
Fructose solution*
Sucrose solution*
Sucralose solution*
Starch solution*
Test tubes
Distilled water
37°C water bath
Invertase Enzyme (Sucrase enzyme)
5% Silver nitrate solution
5% Sodium hydroxide solution
2% Ammonium nitrate solution
Benedict’s Reagent
Iodine solution

* The solutions will be labeled as unknowns A – E.

*************************************Procedure

Gather 5 test tubes and label them A – E. Place about 1 mL of each unknown into the corresponding tube.

Add 1 or 2 drops of iodine solution to each of the tubes and record observations. Presence of a deep blue, purple or black color is a positive test for presence of starch. With the starch identified, you now have 4 unknowns.

Properly dispose of waste, and wash and dry the used test tubes.

Gather 8 test tubes and label them. There will be 2 test tubes for each unknown. Tollen’s test will be performed in one test tube and Benedict’s in the other for each unknown.

Perform Benedict’s and Tollen’s Tests on each of the unknowns and record results.

Tollen’s test: To an empty test tube, add 10 drops of 5% silver nitrate solution and 2 drops of 5% sodium hydroxide. Add enough 2% ammonium hydroxide to just dissolve the black precipitate (shake the tube to mix). Be sure not to add an excess of ammonium hydroxide. Sometimes it is difficult to dissolve all of the precipitate, Add 4 drops of your sugar solution. Formation of a silver mirror on the interior of the test tube is a positive test for a reducing sugar. Gentle heating in the 37°C water bath will facilitate the reaction.

Benedict’s test: In an empty test tube, add 5 mL of the Benedict’s reagent to 2 mL of your sugar solution. Heat these mixtures in a 37°C water bath for 10 minutes. A reducing sugar will produce a red, green or yellow precipitate.

Discard the silvered tubes and clean the remaining test tubes.

The results of Tollen’s and Benedict’s tests should reveal which unknowns are monosaccharides and which are disaccharides. (Is either disaccharide a reducing sugar?) Label two test tubes and place approximately 2 mL of the disaccharide unknowns into the appropriate tube.

Add approximately 2 mg of invertase enzyme to both tubes and place them in the 37 C bath for 20-30 minutes.

While the test tubes are incubating in the bath, polarimetry will be used to identify the monosaccharides. Use the solutions labeled ‘polarimeter A’ and ‘polarimeter B’ for this test. Consult the operating manual and instructor for further details. Record the results.

Specific rotations of fructose and glucose:

fructose  = – 93°glucose  = + 52°

Once the incubation time is complete, repeat Tollen’s test on the disaccharides. A positive test should identify the sucrose solution (now broken down into monosaccharides by the enzyme).

Having completed the above tests, the identity of each unknown should now be established.

***********Postlab Questions

1 a. Draw the products of the acid hydrolysis of lactose.

b. Draw the aldehyde form of lactose.

2. What can you conclude from the results of the Benedict’s and Tollen’s tests? In other words, which sugars are reducing, and which are non-reducing, according to the results of your tests? This answer should be longer than a couple words.

3. Describe, using the polarimetry data, what sugars were generated in test tubes 2 and 3. Please support your claims with evidence!