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Experiment No. 2
Carbohydrates

Carbohydrates are organic substances composed of certain chemical groups,

such as alcohols, aldehydes, or ketone groups. These compounds include natural
sugars which are largely distributed in nature, and are classified as
monosaccharides, disaccharides or polysaccharides based on the complexity of
their molecules.

General Test for Carbohydrates:

1. Molisch Test:
Materials:
Test tubes, test tube racks, pipettes
Reagents:
Molisch Reagent, Concentrated Sulfuric Acid, glucose, fructose, lactose,
Sucrose, starch solution

Procedure:

1. To 1 ml of 0.1 M glucose in a test tube.

2. Add 5 drops of Molisch reagent and shake.
3. Add 1 ml. of concentrated Sulfuric Acid, pouring the acid carefully down the
side of the tube.
4. Note the color formed at the junction of the two layers.
5. Repeat the procedure using 0.1 M sucrose, 0.1 M maltose, 0.1 M fructose, 0.1
lactose and 1% starch solution.

Result:
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-A purple/violet colored ring is observed at the junction of the two liquids which
are molisch test reagent and concentrated Sulfuric Acid.

Sucrose
Glucose
Fructose Carbohydrates are present which
Lactose indicates for positive molisch test
Maltose
Starch solution

Test for Reducing Sugars:

1. Benedict’s Test:
Reagent:
Benedict’s reagent

Procedure:
1. Place 1 ml. of Benedict’s solution in a test tube. Heat to boiling.
2. Add 1 ml. of 0.1 M glucose and continue boiling for 2 minutes.
3. Observe for the change in color.
4. Repeat the procedure using 0.1 M fructose, sucrose, maltose, lactose and 1%
starch solution.

Result:
Glucose- positive result, red precipitate is present
Fructose- positive result, red precipitate is present
Lactose- positive result, red precipitate is present
Maltose- positive result, red precipitate is present
Sucrose- negative result, no precipitate
Starch solution- negative result, no precipitate

2. Nylander’s Test:
Reagent:
Nylander’s reagent

Procedure:
1. Place 1 ml. of 0.1 M glucose and heat in a boiling water bath for 5 minutes
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2. Add 5drops of Nylander’s reagent. Observe the change in color.

3. Repeat the procedure on the other sugars.

Result:

Positive result for Nylander’s reagent Negative result for Nylander’s reagent

Glucose Sucrose
Fructose Starch solution

Lactose
Maltose

3. Barfoed’s Test:
Reagent:
Barfoed’s reagent

Procedure:
1. Place 1 ml of 0.1 M glucose solution in a test tube.
2. Add 1 ml of Barfoed’s reagent
3. Place the test tube in a boiling water bath and note the time required to give a
definite color.
4. Repeat the procedure using the other sugars.

Result:

Sucrose- positive result monosaccharide is present

Lactose- positive result monosaccharide is present
Glucose- negative result disaccharide is present
Fructose- negative result disaccharide is present
Maltose- negative result disaccharide is present
Starch solution- negative result disaccharide is present

NOTE: If a red precipitate is formed within two minutes. A monosaccharide is

present. If the red precipitate is formed after ten minutes of heating, a
disaccharide is present.
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4. Tollen’s Test:
Reagent:
Tollen’s Reagent
Procedure:
1. Place 1 ml of 0.1 M glucose in a test tube.
2. Add 5 drops of freshly prepared Tollen’s reagent.
3. Warm in a water bath for 5 minutes.
4. Allow to stand for 5 minutes. Observe.
5. Repeat the procedure using the other sugars.

Result:
Sucrose- negative result for tollen’s regeant, reducing sugar is absent
Starch solution- negative result for tollen’s regeant reducing sugar is absent
Lactose- positive result for tollen’s regeant, reducing sugar is present
Glucose- positive result for tollen’s regeant, reducing sugar is present
Fructose- positive result for tollen’s regeant, reducing sugar is present
Maltose- positive result for tollen’s regeant, reducing sugar is present

Test for Polysaccharide:

1. Iodine Test:
Reagent:
Iodine Solution

Procedure:
1. To 1 ml of 1% starch solution, and 2 drops of iodine solution.
2. Repeat the procedure using glycogen.
3. To prepare glycogen, get 4 fresh oyster or scallops, cut these into small piece
and drop into vigorously boiling water. Remove the tissue from the water,
grind into fine particles with sand in a mortar, then place in the same water
and boil for 30 minutes. Make faintly acidic acid by adding 3 drops of
concentrated acetic and filter while hot. Use the filtrate for the test.

Result:
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Positive result indicating the presence of starch.