Name: _________________________________ Per:________ Performance: ________
BIOCHEMISTRY LAB # 6
CARBOHYDRATES
BACKGROUND
Carbohydrates are polyhydroxy compounds that are either aldehydes or ketones that contain only
carbon, hydrogen, and oxygen. Carbohydrates comprise about 60% of our daily caloric intake and are
the major source of metabolic energy. Carbohydrates are categorized as monosaccharides,
disaccharides, or polysaccharides.
Monosaccharides:
Are the simplest carbohydrates and serve as the building blocks for di and poly saccharides.
Glucose, galactose, and fructose are examples of monosaccharides. All digestible dietary carbohydrates
in our diet are eventually broken down into glucose, and therefore it is also commonly known as blood
sugar. Foods that tend to elevate blood sugar levels too high and too fast are thought to contribute to
insulin resistance and type 2 diabetes. Fructose is found in fruits and fruit juices and in honey. Fructose
is also commonly known as fruit sugar, and can be found in a large number of foods items (as high
fructose corn syrup).
All monosaccharides are reducing sugars (i,e, they can be oxidized), and either contain an
aldehyde (aldoses) or a ketone (ketoses) group.
Disaccharides:
In a disaccharide two monosaccharides are linked together via a glycosidic bond. Maltose,
lactose, and sucrose are three common disaccharides. Lactose, also commonly known as milk sugar, is
found in milk and milk products. Lactose contains a glucose and galactose combined via a glycosidic
bond. Individuals who lack the enzyme to breakdown (digest) lactose suffer from lactose intolerance.
Sucrose, common table sugar, is composed of glucose and fructose.
Unlike the monosaccharides, not all disaccharides are reducing sugars. Of the three common
disaccharides Lactose and Maltose are reducing sugars, while Sucrose is not.
Polysaccharides:
Polysaccharides are polymers composed of multiple monosaccharide units. Amylose,
Amylopectin, Glycogen, and Cellulose are all polysaccharides containing glucose, and are different only
in the manner in which glucose units are attached to each other. Starch composed of the
polysaccharides amylose and amylopectin, is the major form of glucose storage in plants. Amylose (~
20% of starch) is a linear chain of glucose while amylopectin (~ 80%) is a branched polymer of glucose.
Glycogen is the major form of glucose storage in animals, and is found in the liver and muscle.
Glycogen, like amylopectin in plants, is a branched polymer of glucose. The breakdown (hydrolysis) of
glycogen helps maintain blood glucose levels between meals. Cellulose is an unbranched chain of
glucose and is the major structural component of plants. Humans do not have the enzymes to digest
cellulose and therefore, humans cannot digest cellulose. Such undigestable carbohydrates provide the
major source of fiber in our diet.
Tests for the identification of carbohydrates
Benedict’s test for reducing sugars: Benedict’s reagent reacts with reducing sugars to form a red
precipitate. The color of the precipitate can vary from green to gold to red depending on the amount
(concentration) of the reducing sugar present in the sample.
Seliwanoff’s test for ketoses: Seliwanoff’s test can be used to distinguish 6 carbon monosaccharides
(hexoses) that have a ketone group from those hexoses that contain an aldehyde group. With ketoses
the reagent produces a deep red color rapidly, while with aldoses a light pink color develops over a
longer period of time.
Name: _________________________________ Per:________ Performance: ________
Iodine test for polysaccharides: Iodine interacts with the structure of amylose to produce a deep blueblack complex. The polysaccharides amylopectin, glycogen, and cellulose, interact with iodine to give
red to brown colors. Glycogen gives a reddish-purple color with iodine. Mono and di- saccharides do not
interact with iodine to form dark colored complexes.
HYDROLYSIS OF DISACCHARIDES AND POLYSACCHARIDES
Disaccharides and polysaccharides can be hydrolyzed (broken down), given sufficient time, into their
constituent monosaccharides. In the laboratory hydrolysis can be achieved by reacting the di- and polysaccharides with acid. Acid hydrolysis of disaccharides will produce the constituent monosaccharides.
Acid hydrolysis of polysaccharides will first produce smaller polysaccharides and disaccharides, but
eventually, given sufficient time, will produce the constituent monosaccharides. In our bodies the
hydrolysis of polysaccharides (except cellulose) is carried out by enzymes that are found in our saliva or
secreted from our pancreas.
PROCEDURE
The goal of this laboratory is to introduce you to the concepts related to carbohydrates. You will first
carry out the three identification reactions on samples containing known carbohydrates and record your
observations. Based on these observations you will then attempt to identify the carbohydrate contained
in unknown solutions.
Second, you will hydrolyze a carbohydrate using acid. Once the carbohydrate has been treated with acid
you will test for hydrolysis of the sample using the Benedict’s test and the iodine test. You will compare
the samples with acid to samples that did not contain any acid to determine if the carbohydrate was
hydrolyzed.
Finally you will test a common food item for the presence of different carbohydrates using the three tests
you performed in the first part.
Materials: One large beaker (250-400 mL), plastic well plate, micro-centrifuge tubes and clips, boiling
water bath, floating test tube rack, hot plate, beral pipettes, and plastic 24-well plate.
A. BENEDICT’S TEST FOR REDUCING SUGARS
Materials: Benedict’s reagent and 2% carbohydrate solutions: glucose, fructose, sucrose, lactose, 1%
starch and unknown.
Place 5-6 drops of solutions of glucose, fructose, sucrose, lactose, starch, water and an unknown in
separate, labeled micro-centrifuge tubes. Add 9 drops of Benedict’s reagent to each sample, then cap
and gently shake the closed tube to mix. Heat all the tubes in a boiling water bath for 3-4 minutes. The
formation of a greenish to reddish-orange color indicates the presence of a reducing sugar. If the
solution is the same color as the Benedict’s reagent in water (the control), then there is no oxidation
reaction. Record your observations. Classify each as reducing or non-reducing sugar.
B. Seliwanoff’s Test for Ketoses
Materials: Seliwanoff’s reagent and 2% carbohydrate solutions: glucose, fructose, sucrose, lactose, 1%
starch and unknown.
Place 2-3 drops of solutions of glucose, fructose, sucrose, lactose, starch, water and unknown in
separate, labeled micro-centrifuge tubes. Add 10 drops of Seliwanoff’s reagent to each sample, then cap
and gently shake the closed tube to mix. The reagent contains concentrated HCl. Use carefully.
Name: _________________________________ Per:________ Performance: ________
Place the test tubes in a boiling hot water bath and note the time. After 5 minutes, observe the colors in
the tube. The formation of a deep red color indicates the presence of a ketose. Record your results as a
deep red color change, pale red/pink change or no change.
C. IODINE TEST FOR POLYSACCHARIDES
Materials: Iodine reagent and 2% carbohydrate solutions: glucose, fructose, sucrose, lactose, 1%
starch and unknown.
Place 5-6 drops of each solution of glucose, fructose, sucrose, lactose, starch, water and an unknown in
separate wells in your well plate. Add 1 drop of iodine reagent to each. A dark blue-black color is a
positive test for amylose in starch. Record your results. Complete the table to identify the carbohydrates
in your unknown.
D. HYDROLYSIS OF DISACCHARIDES AND POLYSACCHARIDES
Materials: 10% HCl, 10% NaOH, red litmus paper, iodine reagent, Benedict’s reagent, 1% starch and
2% sucrose solutions, stir rod.
Place 7-8 drops of 1% starch in two micro-centrifuge tubes and 7-8 drops of sucrose in two more microcentrifuge tubes. To one sample of sucrose and starch, add 3-4 drops of 10% HCl. To the other
samples of sucrose and starch, add 3-4 drops of DI water. Label the test tubes and gently shake the
closed tubes to mix, and then heat in a boiling water bath for 10 minutes.
Remove the test tubes from the water bath and let them cool. To the samples containing HCl, add 10%
NaOH (about 3-4 drops) and shake the closed tube to mix. Add NaOH until the solution turns red litmus
paper blue, indicating the HCl has been neutralized. Apply a drop of solution from the tip of the stir rod
onto the litmus paper to check pH. Test the samples for hydrolysis as follows:
Iodine Test: Place 5-6 drops of each solution in a well and add 1 drop of iodine solution to each. Add
extra iodine solution if the color disappears. Continue to add iodine solution one drop at a time until a
permanent color is obtained. Record your observations. Determine if hydrolysis has occurred in each.
Benedict’s Test: Add 9 drops of Benedict’s reagent to each of the remaining samples and heat in a
boiling water bath for 3-4 minutes. Determine if hydrolysis has occurred in each.
E. TESTING FOODS FOR CARBOHYDRATES
Materials: Obtain a carbohydrate sample to test. Perform Benedict’s, Seliwanoff’s and iodine tests on
each. Describe the kinds of carbohydrates you identify in each sample.
PRE-LABORATORY QUESTIONS- 2PTS EACH
Read the lab procedures, then answer the pre-lab questions on a separate sheet of paper. Points will be
taken off for sloppy work…
1. Draw the reaction of glucose with the Benedict’s reagent.
2. Draw the hydrolysis reaction of sucrose, structures and names.
3. Based on your knowledge about the different tests for Carbohydrates, predict the outcome (final
color) of the Benedicts test and the Seliwanoff’s test with Sucrose, before and after hydrolysis.
4. List three examples each for monosaccharides, disaccharides and polysaccharides. For the di- and
poly- saccharides also list its constituent monosaccharide.
5. Write the formula for sucrose, maltose and lactose
REPORT SHEET
A. BENEDICT’S, B. SELIWANOFF’S AND C. IODINE TESTS FOR CARBOHYDRATES
Compound
Observations
A. Benedict’s
Glucose
Fructose
Sucrose
Lactose
Starch
Water
Unknown #_________
B. Seliwanoff’s
C. Iodine
D. HYDROLYSIS OF DISACCHARIDES AND POLYSACCHARIDES
Test
Observations
Sucrose + H2O
Sucrose + HCl
Starch + H2O
Starch + HCl
Iodine
Benedict’s
E. TESTING FOODS FOR CARBOHYDRATES
Food sample tested: _______________________________________
Test
Observations
Benedict’s
Seliwanoff’s
Iodine
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QUESTIONS
1. In parts A, B, and C you performed three tests to identify various carbohydrates. Based on your
observations for the known solutions and your observations for the unknown solution, what
carbohydrate(s) is/are in your unknown solution? Provide a brief explanation as to how you made
your choice.
2. Which sucrose and starch samples in part D undergo hydrolysis? How do the observations for the
iodine and Benedict’s tests indicate that hydrolysis has occurred?
3. Based on your observations in part D what are the hydrolysis products for each tube:
Sucrose + H2O
Sucrose + HCl
Starch + H2O
Starch + HCl
4. Based on your observations in part E what possible carbohydrates are present in the food item you
tested?
5. You have in front of you three vials containing a clear liquid. You are told that each vial contains one
of the following carbohydrates: Fructose, Sucrose, Amylose. What experiments could you carry out
to determine the composition of each vial. Write down the experiment(s) as well as the expected
observations.
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