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 cyclic hemiacetal 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.
1. List the hazardous chemicals we will be working with.
2. What function does the invertase enzyme perform?
3. 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
1. Gather 5 test tubes and label them A – E. Place about 1 mL of each
unknown into the corresponding tube.
2. 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.
3. Properly dispose of waste, and wash and dry the used test tubes.
4. 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.
5. 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.
6. Discard the silvered tubes and clean the remaining test tubes.
7. 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.
8. Add approximately 2 mg of invertase enzyme to both tubes and place
them in the 37 C bath for 20-30 minutes.
9. 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°
10. 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!