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ACTIVITY 6

Carbohydrates

ASA
6

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Pre-laboratory Questions

1. Describe the differences among monosaccharides, disaccharides and polysaccharides.


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2. Identify each of the following as a) aldose or ketose b) a triose, tetrose, pentose, hexose or heptose.
b)

c)

d)

CH2OH

CH2OH

C=O

C =O

C=O

C=O

H C OH

HO C H

HO C H

H C OH

H C OH

H C OH

H C OH

H C OH

H C OH

CH2OH

CH2OH

CH2OH

Aldose or ketose
Figure a
Figure b
Figure c
Figure d

H C OH
CH2OH

Triose, tetrose, pentose, hexose or heptose

ACTIVITY
6

Carbohydrates

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Group

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Date

ACTIVITY 5
Carbohydrates
INTRODUCTION
Carbohydrates are polyhydroxy aldehydes, polyhydroxy ketones, or substances that by simple
hydrolysis yield these compounds. Carbohydrates that cannot be hydrolyzed to simpler molecules are called
monosaccharides or simple sugars. Examples are glucose, fructose and galactose. Disaccharides can be
hydrolyzed to two monosaccharides. Sucrose, maltose, and lactose are common examples. Polysaccharides
such as starch and cellulose are polymers and are hydrolyzed to several molecules of monosaccharides.
Carbohydrates are classified further as being either reducing or non-reducing sugars. Reducing
carbohydrates contain free or potential aldehyde or ketone group in the hemiacetal type structure and reduce
+
2+
+
Ag to metallic silver or Cu to Cu . All monosaccharides and nearly all disaccharides are reducing sugars.
Sucrose is not, and neither are the polysaccharides.
Reactions with phenylhydrazone and with strong oxidizing agents, such as concentrated HNO 3 are
useful in carbohydrate identification. Crystalline substances called osazones and dicarboxylic acids are formed
respectively.
Tests and Reactions
Molisch test is a general test for carbohydrates, and monosaccharides will give a positive test rapidly.
Disaccahrides and polysaccharides will slowly hydrolyse to produce monosaccharides in the acidic medium of
the test and will also give a positive test.
The Benedicts test is used to detect the presence of reducing sugars. Any monossacharide or
disaccharide that can form a free aldehyde group in solution will react with Benedicts reagent to form a brickred, brown, green or occasionally yellow precipitate. A colored solution with no precipitate is a negative result.
The Seliwanoff test will distinguish between ketohexoses and aldohexoses. The difference in the times
required for the red color to appear is used to distinguish ketohexoses from aldohexoses.
The Barfoeds test is used to distinguish between reducing monosaccharides and reducing
disaccharides. Its color reaction is similar to Benedicts reagent.
The iodine test is used to detect the presence of polysaccharides. Starch will borm a dark-blue complex
with iodine.
The Bial test differentiates pentoses from hexoses. A blue color is positive test for a pentose. All other
colors are considered negative for pentoses.
OBJECTIVES
To observe general and specific reactions of monosaccharides, disaccharides and polysaccharides.
To identify an unknown sample of carbohydrates using their properties
MATERIALS
test tube (12), test tube holder, test tube rack, dropper, 5-mL pipet, 600-mL beaker, wire gauze, tripod, burner,
evaporating dish, cork (6)
REAGENTS
5% glucose, 5% fructose, 5% sucrose, 5% galactose, 5% maltose, 5% lactose, distilled water, Molisch reagent,
Benedicts reagent, Barfoeds reagent, Nylanders reagent, Seliwanoff reagent, concentrated H 2SO4,
concentrated HNO3, 0.1M iodine-KI solution, phenylhydrazine HCl, sodium acetate

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PROCEDURE
I. General Classification Test
A. Molisch Test
1. Place 2 ml of 5% glucose solution in a test tube. Then add 2 drops of Molisch reagent. Mix well.
2. Incline the tube and carefully pour down the side 2 ml of concentrated H 2SO4. Avoid mixing of
the two layers.
3. Note the color formed at the junction of the layers.
4. Repeat the test using starch solution, then sucrose in place of glucose solution.
II. Tests Depending on the Ability of Carbohydrates to Reduce Metals
1. Place 1 ml each of 5% glucose, fructose, galactose, maltose, lactose, and sucrose separately
into 6 test tubes.
2. Add 2 ml of Benedictss reagent. Mix well.
3. Place in boiling water bath for at least 20 mins.
4. Note any change in color and formation of precipitate.
5. Repeat the procedure using Barfoedss instead of Benedicts reagent. This time record the
time required for an observable change in each tube.
6. Repeat steps 1 to 4, this time using Nylanders reagent in place of Benedicts. Tabulate the
results.
III. Specific Tests for Carbohydrates
A. Seliwanoffs test
1. Place 1 mL each of the sugar solutions separately into 6 test tubes.
2. Add 2 mL of Seliwanoffs reagent. Mix well
3. Place in boiling water bath.
4. Record the time required for any change in color to occur. Leave the tubes in the water bath
for 10 mins. before recording negative results.
B. Galactaric (Mucic) acid test
1. Place 0.5 g each of lactose, galactose and glucose separately into 3 test tubes.
2. To each tube add 1 mL of water and 1 ml of concentrated HNO3. Mix until solids dissolve.
3. Place the test tubes in boiling water bath for 1 to 1-1/2 hours. Remove the tubes from the
bath and allow to stand overnight. Examine the presence of crystals.
C. Iodine test
1. In an evaporating dish, add a drop of starch solution to a drop of 0.1 M iodine in KI solution.
2. Repeat the test using glucose, then sucrose solution, in place of starch.
3. Record your results.
IV. Identification of Sugars by Osazone Formation
1. Place approximately 1 gram of phenylhydrazine mixture (2 parts phenylhydrazine hydrochloride
and 3 parts sodium acetate by weight) into each of the 6 test tubes.
2. Add 3 mL each glucose, fructose, galactose, maltose, lactose, and sucrose solutions separately into
the 6 test tube. Shake vigorously to insure complete solution.
3. Stopper the mouth of the tubes loosely with cotton and place in a boiling water bath. Record the
time.
4. Observe. As soon as a distinct precipitate is formed, record the time and remove the tube from the
bath.
5. If no precipitate forms after 40 mins., remove the tube from the bath and 3 ml of water. The cooling
effect of added water usually causes an immediate precipitation of the osazones.
6. Examine and draw the crystals seen under the microscope.

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V. Identification of an unknown Carbohydrate


NOTE: Refer to procedures and visible results obtained in parts I-IV of this experiment.
Obtain an unknown solid carbohydrate (or unknown mixture) from your laboratory instructor.
Prepare a solution that contains approximately 5% of your unknown by dissolving 0.5 g in 10 ml of
water.
Using a drop of the prepared unknown solution perform iodine test. If the result is positive, starch is
present. If the result is negative, starch is absent.
Perform Benedicts test using 1 mL sample of the unknown.
If this test is negative, it indicates the absence of reducing sugars and the possible presence of sucrose.
The presence of sucrose may be confirmed by testing 1 ml of the unknown solution with
Seliwanoffs reagent.
If Benedicts test is positive, reducing sugars are present. However, if the unknown is a mixture, a
positive Benedicts test does not mean sucrose is absent. Test the presence of sucrose, using
Seliwanoffs test.
Perform Barfoeds test on another 1 ml sample of the unknown solution.
If no brick-red precipitate appears in 5 minutes, this indicates absence of monosaccharides. The
unknown may be tested for the presence of disaccharides, lactose and maltose. To do this,
perform fermentation test or mucic acid test.
If a brick-red precipitate appears in 5 minutes, test for the presence of monosaccharides. Test for the
presence of monosacharides. Test for the presence of fructose by performing Seliwanoffs test. Test
also for the presence of glucose and galactose by performing either fermentation test or mucic acid
test.
If the unknown is a mixture, appearance of brick red precipitate does not mean disaccharides are
absent.

PRE-LAB
6

Carbohydrates

Name

Group

Section

Schedule

Instructor

Date

I. Objectives
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II. Materials and Reagents
A. Materials
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B. Reagents
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III. Procedure

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DATA AND RESULTS


I.

General Classification Test


A. Molisch test
Test Solutions

Observations

Glucose
Sucrose
Starch
B. Fermentation test
Test Solutions

Observations

Glucose
Sucrose
Lactose
Starch
II. Tests Depending on the Ability of Carbohydrate to Reduce Metals
Test Solutions
Benedicts

Barfoed

Glucose
Fructose
Galactose
Maltose
Sucrose
Lactose
III. Specific Tests for Carbohydrates
A. Seliwanoffs
Test Solutions
Glucose
Fructose
Galactose
Maltose
Sucrose
Lactose

Observation

Nylanders

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B. Mucic Acid Test


Test Solutions

Observation

Glucose
Galactose
Lactose

C. Iodine Test
Test Solutions

Observation

Glucose
Sucrose
Starch
IV. Identification of Sugars by Osazone Formation
Sugar Tested
Initial Time

Precipitation Time

Illustration

Glucose
Fructose
Galactose
Lactose
Maltose
Sucrose
V. Identification of Unknown Carbohydrate
Unknown Number: ___________
Tests performed

Results

Identity of unknown: _______________

Interpretation

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IV. DISCUSSION OF RESULTS


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V. CONCLUSION
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QUESTIONS TO ANSWER
1.

Tell what is determined by each of the following:

a. a positive Molisch test ___________________________________________________________________


b. a positive Benedicts test _________________________________________________________________
c.

a positive Seliwanoffs test ________________________________________________________________

d. formation of a brick-red precipitate within 3 to 5 mins. in Barfoeds test


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2. Tell how you would use a single test to distinguish between each of the following pairs of substances. Give
both the test and results expected with each substance.
a. Starch
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b. sucrose and maltose
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c.

maltose and lactose

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3. Why is sucrose non-reducing with Benedicts test while lactose is a reducing sugar under the same
conditions?
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4. Why is sucrose fermented by yeast while lactose is not?
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5. Why does a monosaccharide react more rapidly with Barfoeds than a reducing disaccharide?
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