MOLISCH’S TEST

1. Reagent

 Molisch reagent: Dissolve 3.75 g of α-naphthol in 25 ml of Ethanol 99%. This reagent should
be prepared fresh.

 Concentrated sulphuric acid

 Test sample

2. Materials required

 Test tubes

 Test tube stand

 Pipette

 Distilled water

Procedure of Molisch Test

1. Take 2 mL of each distilled water and test sugar solutions in four test tubes separately.

2. Add two drops of Molisch reagent to each tube.

3. Hold the test tube in an inclined position and gently add 1 mL concentrated H2SO4 along the
wall of the test tube. Do not mix the acid with the solution. A black ring may form if
concentrated acid is not added slowly as the heat generated from the reaction can char the
carbohydrates.

4. Observe the test tube for the formation of a purple-colored ring at the layer between the
solution and the acid.

Result and Interpretation of Molisch Test

  The formation of the purple colored ring occurs at the interface between the sulphuric acid
and the test solution.

 The sulphuric acid remains above the test solution as the acid is denser than the test
solution.

 The absence of color indicates a negative result.

Uses of Molisch Test

 Molisch test is used to detect the presence of carbohydrates in different samples.

 It can be used to detect the formation of carbohydrates as a by-product in different reactions

and distinguish it from other biomolecules.

FEHLING’S TEST

1. Reagent

 Fehling’s solution A: Dissolve 7 g of CuSO4.7H2O in 100 mL of water.

 Fehlings solution B: Dissolve 24 g of KOH and 34.6 g of potassium sodium tartrate in 100 mL
water.

 Fehling’s solution: Mix equal volumes of both the solution just before use.

 Sample (5% Glucose, 5% Sucrose, 5% Fructose, 5% Starch, 5% lactose)

2. Materials Required

 Pipettes

 Test tubes

 Test tube stand

3. Equipment

 Water bath

Procedure of Fehling’s Test

1. Take 1 mL of a given sample in a clean, dry test tube. The concentration of the test samples
should be 5% (w/v).

2. Take control of 1 mL of distilled water in another tube.

3. Add about 2-3 drops of Fehling’s reagent to both the tubes and mix them in a vortex.

4. Keep the test tubes in the water bath for 1-2 minutes.

5. Observe the appearance of color in the test tubes.

6. Note down the appearance of color seen in the test tubes.

Result and Interpretation of Fehling’s Test

 The appearance of a reddish-brown precipitate indicates a positive result and the presence
of reducing sugars.

 The absence of the reddish precipitate or the appearance of deep blue color indicates a
negative result and lack of reducing sugars.

Uses of Fehling’s Test

 Fehling’s test is used to distinguish between the presence of aldehydes and ketones in
carbohydrates as ketone sugars except alpha-hydroxy-ketone do not react in this test.

 Fehling’s test is performed in medical facilities to detect the presence of glucose in urine.
This helps to identify whether the patient has diabetes or not.

BENEDICT’S TEST

Requirements of Benedict’s Test

 Sample solution of unknown carbohydrate (or urine sample)

 Test-tubes and test-tube holders

 Pipette

 Bunsen burner

 Benedict’s Reagent

Preparation of Benedict’s Reagent

1. Measure 17.3 grams of copper sulfate (CuSO4), 173 grams of sodium citrate (Na3C6H5O7), and
100 grams of anhydrous sodium carbonate (Na2CO3) (or 270 grams of sodium carbonate
decahydrate (Na2CO3.10H2O))

2. Put all the measured chemicals in a volumetric flask of 1000 mL.

 3. Pour distilled water up to 1000 mL marking.

4. Dissolve all the components properly by shaking gently.

Figure- Preparation of Benedict’s Reagent.

Procedure of Benedict’s Test

1. In a clean test tube add 1 mL of sample solution (urine or carbohydrate solution).

2. Add 2 mL of Benedict’s reagents over the sample.

3. Place the test tube over a boiling water bath and heat for 3–5 minutes or directly heat over a
flame.

4. Observe for color change.

Figure- Procedure of Benedict’s Test.

Result Interpretation / Observation of Benedict’s Test

Any change in color from blue to green or yellow or orange or red within 3 minutes indicates a
positive Benedict test i.e. presence of reducing sugar in the sample.
For semiquantitative evaluation, the concentration of reducing sugar can be estimated based on the
shade of developed color as follows;

Approx. Concentration of
Shade of Color Indication
Reducing Sugar (in g%)

Blue 0 No reducing sugar

Green solution < 0.5 Trace reducing sugar

Green ppt. 0.5 – 1 Trace reducing sugar

Yellow ppt. 1 -1.5 Low reducing sugar

Orange-red ppt. 1.5 – 2 Moderate reducing sugar

Brick-red ppt. >2 High reducing sugar

Figure- Result Interpretation, Observation of Benedict’s Test.

BARFOED’S TEST

1. Reagent

 Barfoed’s reagent: 0.33M solution of copper acetate is added to 1% acetic acid. The freshly
prepared reagent should be used for the assay.

 Sample
2. Materials Required

 Test tubes

 Test tube stand

 Pipettes

3. Equipment

 Water bath

 Vortex

Procedure of Barfoed’s Test

1. Take 1 mL of a given sample in a clean, dry test tube. The concentration of disaccharides
sample (if used) should not exceed 1% (w/v).

2. Take control of 1 mL of distilled water in another tube.

3. Add about 2-3 drops of Barfoed’s reagent to both the tubes and mix them in a vortex.

4. Keep the test tubes in the water bath for 1-2 minutes. The boiling should not be done for
more than 2 minutes as the disaccharides might hydrolyze into monosaccharides and give a
positive result.

5. Observe the appearance of color in the test tubes.

6. Noted own the time taken for the appearance of color in the tubes.

Result and Interpretation of Barfoed’s Test

 The presence of red precipitate detects the presence of reducing monosaccharides in the
sample.

 If the color appears within the first few minutes, the sample contains reducing
monosaccharides.
  However, if the color appears later than the first 3 minutes, the sample is of reducing
disaccharides.

Uses of Barfoed’s Test

 This test is used to identify reducing monosaccharides and distinguish the reducing
disaccharides from reducing monosaccharides.

IODINE’S TEST

1. Reagent

 Lugol’s iodine: 5% elemental iodine is mixed with 10% potassium iodide to form the Lugol’s
iodine.

 Test sample

2. Materials Required

 Test tubes

 Test tube stand

3. Equipment

 Water bath

Procedure of Iodine Test

1. Take 1 mL of a given sample in a clean, dry test tube.

2. Take control of 1 mL of distilled water in another tube.

3. Add about 2-3 drops of Lugol’s solution to both the tubes and mix them in a vortex.

4. Observe the appearance of color in the test tubes.

5. Heat the test tubes in the water bath until the color disappears.

6. Take the test tubes out for cooling

7. Note down the appearance of color seen in the test tubes.

Result and Interpretation of Iodine Test

  The appearance of blue-black or purple color represents a positive test, indicating the
presence of starch.

 If there is no change in color, the result is negative and indicates the absence of starch.

Uses of Iodine Test

 This test is used to detect the presence of starch in various samples.

 Similarly, this test is performed to test the process of photosynthesis in plants.