Laboratory Exercise # 12

QUALITATIVE ANALYSIS OF SUGARS

INTRODUCTION:

Sugar is the generalised name for a class of chemically-related sweet-flavored substances, most of which are used as food.
They are carbohydrates, composed of carbon, hydrogen and oxygen. There are various types of sugar derived from
different sources. Simple sugars are called monosaccharides and include glucose (also known as dextrose), fructose
and galactose.

The table or granulated sugar most customarily used as food is sucrose, a disaccharide (in the body, sucrose hydrolyses
into fructose and glucose). Other disaccharides include maltose and lactose. Chemically-different substances may also
have a sweet taste, but are not classified as sugars. Some are used as lower-calorie food substitutes for sugar described
as artificial sweeteners.

Several qualitative tests have been devised to detect members of this biologically significant class of compounds. These
tests will utilize a test reagent that will yield a color change after reacting with specific functional groups of the compounds
being tested. The following exercises are reactions that can detect the presence or absence of carbohydrates in test
solutions. They range in specificity to the very general (i.e., Molisch test for carbohydrates) to the very specific (i.e., mucic
acid test for galactose).

1. Molisch’s Test for Carbohydrates

2. Benedict’s Test for Reducing Sugars
3. Barfoed’s Test for Monosaccharides
4. Lasker and Enkelwitz Test for Ketoses
5. Bial’s Test for Pentoses
6. Mucic Acid Test for Galactose
7. Iodine Test for Starch and Glycogen

LEARNING OUTCOMES:

By the end of this exercise, you should be able to:

 identify the characteristics of carbohydrates;
 define what is a reducing sugar;
 Differentiate between monosaccharides and disaccharides in terms of their reactions

Materials:

 Molisch Reagent 1 % solutions of: Filter paper

 Benedicts solution Glucose Glasswares
 Barfoed’s reagent Fructose
 Bial’s Orcinal reagent Lactose
 Seliwanoff’s reagent Sucrose
 Iodine solution Starch
 Concentrated sulfuric acid
PROCEDURE:

Molisch Test (positive test shows a purple condensation product)

1. Add 2 drops of Molisch reagent to 2 mL of 1% glucose solution and mix.

2. Incline the tube and pour slowly and carefully about 3 mL of concentrated sulfuric acid down the side to form a layer
below the sugar solution.
3. Observe for the result.
4. Repeat the test using 1% solution of sucrose and ANOTHER test using 1% solution of starch.
5. Observe for the results.
6. Complete the table in the reports and answer the questions.

Benedict’s Test (Test for reducing sugar: Positive result shows a red precipitate)

1. Prepare 7 test tubes and add the following:

Tube 1: 5 drops of glucose +1.5 ml of water (D1)
Tube 2: 5 drops of dilution from tube 1 + 1.5 ml of water (D2)
Tube 3: 5 drops of dilution from tube 2 + 1.5 ml of water (D3)
Tube 4: 5 drops of dilution from tube 3 + 1.5 ml of water (D4)
Tube 5: 5 drops of dilution from tube 4 + 1.5 ml of water (D5)
Tube 6: 8 drops of sucrose
Tube 7: 8 drops of starch
2. To each prepared tubes add 2.5 mL of Benedict’s reagent and mix the contents.
3. Place the tubes in a boiling water bath for three minutes, then cool the tubes and compare them.
4. Observe and record the results.

Barfoed’s Test (Test to distinguish monosaccharides and disaccharides: Positive result is a red precipitate)

1. Prepare 3 test tubes and 3 mL of Barfoed’s reagent.

2. Add 2 mL of each of the 1% sugar solutions (tube 1-glucose; tube 2-sucrose; tube 3-starch). Mix and place tubes in
a boiling water bath.
3. Observe the tubes during the first five minutes and remove any tube that becomes cloudy or changes color, noting
the time of reaction.
4. Continue heating the tubes for another five to ten minutes, noting the time of reaction in each tube.
5. Record results.

Bial’s Orcinol Test (Test for furfurals: positive result is a blue condensation product)

1. Prepare 3 test tubes and add 3 mL of Bial’s orcinol reagent

2. Add 0.5 mL of each of the 1% sugar solutions (tube 1-xylose; tube 2-glucose; tube 3-lactose). Mix and place the
tubes in a boiling water bath.
3. Observe until a color develops. Record the time of color development. Answer the questions in the report.

Seliwanoff’s Test (Test for ketohexose versus aldohexose: Fructose - deep red color; glucose - light pink color)

1. Add 3 mL of Seliwanoff’s reagent in 4 test tubes.

2. Add 3 drops of 1 % solutions of each sugar (tube 1-fructose; tube 2-glucose; tube 3-sucrose; tube 4-starch). Mix
and place in a boiling water bath.
3. Heat for 10 minutes. Record the time of color formation in each tube.
Iodine Test (Test for Starch)

1. Prepare 3 tubes and add 2 mL of each 1% sugar solutions (tube 1-glucose; tube 2-sucrose; tube 3-starch).
2. Add a few drops of iodine solution.
3. Observe for color changes. Record.
4. Heat the tube containing starch and iodine for as long as five (5) minutes. Observe what happens.
 Laboratory Exercise # 12
QUALITATIVE ANALYSIS OF SUGARS

WORKSHEET

Name: ______________________________ Date :_________

Year and Section: _____________________ Group No: _____

OBSERVATIONS AND GUIDE QUESTIONS.

Molisch Test (positive test shows a purple condensation product)

Sugar Observation Result for Molisch Test
D1 of glucose
D2 of glucose
D3 of glucose
D4 of glucose
D5 of glucose
Sucrose
Starch

1. Why do monosaccharides, oligosaccharides and polysaccharides all give a positive Molisch test?
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2. Will a deoxy-ribonucleic acid give a positive Molisch test? Explain.

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Benedict’s Test (Test for reducing sugar: Positive result shows a red precipitate)

Result for Benedict’s

Sugar Observation
Test
Glucose
Sucrose
Starch
 3. Which sugars reduce the reagent?
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4. Give a definition of a reducing sugar.

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Barfoed’s Test (Test to distinguish monosaccharides and disaccharides: Positive result is a red precipitate)

Sugar Time of Observation Result for

reaction Barfoed’s Test
Glucose
Sucrose
Starch

5. Which sugars gave a positive test within 5 minutes? What type of sugars are these?
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6. What are the average times of reaction of the monosaccharides versus the disaccharides?
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Bial’s Orcinol Test (Test for furfurals: positive result is a blue condensation product)

Time of Result for Bial’s

Sugar Observation
Reaction Orcinol Test
Xylose
Glucose
Lactose
 7. What type of carbohydrate rapidly reacts with the orcinol reagent?
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8. What compound formed in the test reacts with the orcinol to produce a color?
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Seliwanoff’s Test (Test for ketohexose versus aldohexose: Fructose - deep red color; glucose - light pink color)

Time of Result for

Sugar Observation
Reaction Seliwanoff’s Test
Fructose
Glucose
Sucrose
Starch

9. What type of carbohydrate reacts most rapidly in this test?

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10. Upon long heating, a solution of glucose gives a color in the test. Explain why.
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11. How could this test be used to distinguish fructose from sucrose?
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Iodine Test (Test for Starch)

Sugar Observation Result for Iodine Test

Glucose
Sucrose
Starch

12. What happens to starch and iodine after heating?

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13. Explain how might heating caused the change in the starch-iodine complex.
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Summary and Conclusion:

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References:
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