Iodine Test – Iodine test for starch Principle, Procedure, Result, Uses

What is iodine test/Iodine test for carbohydrate?

Some polysaccharides, such as amylase, dextrin, and glycogen, can be distinguished from mono- or disaccharides by a chemical test called the iodine test. An alternative to this procedure, called the starch-iodine test, is used to detect glucose production in plants through their leaves.

Objectives of Iodine Test

  • To detect the existence of polysaccharides, primarily starch.

Principle of Iodine Test

  • Polyiodide ions form colourful adsorption complexes with helical chains of glucose residue in amylase (blue-black), dextrin (black), or glycogen (red) (reddish-brown).
  • Cellulose and other branching polysaccharides, like monosaccharides and disaccharides, are still colourless. The pigment amylopectin produces is an orange-yellow.
  • Lugol’s iodine is an aqueous solution of elemental iodine and potassium iodide that is used as the reagent in the iodine test.
  • By itself, iodine does not dissolve in water. Iodine ions react with each other to produce triiodide ions upon addition of potassium iodine; these ions then react with an iodine molecule to produce pentaiodide ions.
  • The iodide, triiodide, and pentaiodide ions are colourless, whereas the bench iodine solution is brown.
  • It has been found that the helix (coil or spring) shape of the glucose chain is crucial to the success of the experiment.
  • Even more, the length of the glucose chains determines the final hue.
  • The resulting triiodide and pentaiodide ions are linear and can diffuse freely within the helix.
  • It is suggested that the complex’s hue arises from a shift in the gap between energy levels brought about by charge transfer between the helix and the polyiodide ions.
  • As the temperature rises and organic molecules like ethanol, which are miscible in water, are present, the colour intensity fades.
  • When heated, the helical structure of amylose is disturbed, causing it to no longer bind iodine and so lose its blue hue. However, the iodine-binding capacity of amylose is restored when it cools.
  • After being cooled, the blue hue returns because the helical structure is restored, allowing it to once again bind iodine.

Difference between Amylose & Amylopectin

Straight chain polymer comprised of D-glucose subunitsThe polymer is a branched-chain one made of D-glucose subunits
In Amylose the sugar units are linked via glycosidic linkages of a-1,4.In Amylopectin the glucose units are linked by the glycosidic linkage of A-1,6
20 percent of starch are amylose80percent of starch is amylopectin, which is the main ingredient in starch.
Amylose, which is a water insoluble portion of starch.Amylopectin is the water-soluble component of starch.
It does not show signs of gelling upon the addition of hot water.Gelling is evident upon the introduction of hot water
It produces blue color using the iodine solution.It is not able to make blue with the iodine solution


  • Reagent: Lugol’s iodine, 5% elemental iodine is mixed with 10% potassium iodide to form the Lugol’s iodine.
  • Materials Required: Test tubes, Test tube stand
  • Equipment: Water bath

Procedure of Iodine Test

  1. Gather a 1 millilitre sample in a dry, clean tube.
  2. Manage 1 ml of sterile water in a separate tube.
  3. Vortex the two tubes together after adding two or three drops of Lugol’s solution.
  4. Look at the test tubes and see what colours develop.
  5. In a water bath, heat the test tubes until the colour vanishes.
  6. Remove the test tubes from the oven to cool them down.
  7. Observe the test tubes and write down what colours you notice.
Procedure of Iodine Test
iodine test for starch diagram | Source: Dr. Amita Joshi of Biology Online.

Result and Interpretation of Iodine Test

  • A positive test is when a purple or blue-black color appears. This indicates the presence of starch.
  • If the color does not change, it is negative. This indicates that there is no starch.
Result and Interpretation of Iodine Test
Result and Interpretation of Iodine Test

Iodine test for starch in leaves


  • Sample plants.
  • a glass jar or beaker to put the leaves of the plant in.
  • To boil water, you need a stove and a pot.
  • Iodine solution is used to see if there is starch.
  • Ethyl alcohol to break down chlorophyll.
  • Pick-up tools for leaves.

Procedure for Iodine test for starch in leaves

  1. Grow one plant in a room with no light and another in a room with normal light.
  2. Take a beaker with ethyl alcohol in it and a pan with water in it.
  3. After 24 hours, put the beaker of ethanol in the saucepan and heat it until it starts to boil.
  4. Holding the leaves with the tweezers, put them in the boiling water for about 2 minutes.
  5. Then, put the leaves in the ethanol until they stop being green.
  6. Then, put the leaf in a shallow dish and let it cool down.
  7. Put a few drops of iodine on it to find out if it has starch.


It was discovered that the leaf kept in sunlight turns blue-black as starch is produced, whereas the plant kept in darkness turns reddish-brown, indicating that no starch is being produced.


Uses of Iodine Test/What is the purpose of the iodine test? Why is it an essential laboratory procedure?

  • Iodine test is used to detect starch in supplied Samples.
  • Iodine solution is used to test for differentiating starch from monosaccharides, disaccharides, and other polysaccharides.
  • Starch, glycogen, and other carbs can be differentiated using the iodine test.
  • Patients with hyper- or hypothyroidism can be identified through blood iodine testing.
  • The starch hydrolysis test operates on the same premise as the iodine test for starch.
  • Based on the iodine test, iodometric titrations include a starch indicator.

Limitations of Iodine Test

  • The iodine test has a major drawback: it is not quantitative. This means that the test can be used to detect starch content in the sample. The iodine test cannot be used to estimate the amount of starch in the sample.
  • Another limitation is starch hydrolysis under acidic conditions. Acidic samples will therefore not pass the iodine test.
  • A very dark-colored sample cannot be tested for iodine as it will not detect color changes.

Important Points to Remember

  • Lugol’s Iodine Solution is sensitive to light. It should be kept in dark bottles in a dark area.
  • The iodine testing is only for starch. Lugol’s solution of iodine will not cause cellulose to change in color.
  • Temperature changes are not a problem for the test.

The chemistry behind the Iodine test

The basic principle of the iodine testing is that Amylose reacts with starch to form a blue-black complex with the Iodine.

Amylose’s helical structure forms a charge transfer complex (CT) with iodine. Iodine is found within the spiral or helical structure. This is why iodine in water (i.e. an aqueous solution molecular iodine I (I) or potassium iodide KI), is used for the test. It is also known as Lugol’s iodine. This is also known as IKI solution.


I + KI = IKI solution

Let’s now understand Lugol’s iodine purpose.


Water is insoluble for Molecular Iodine, or the iodine molecular, also known as the I2 molecule. For making laboratory reagents, potassium iodide can be used.

Potassium-iodide is dissociated to form iodide ions. Triiodide Ions are formed when iodide and iodide elements combine to form I3-, which then associate to make In- (polyiodide) in solution. I3-chemistry is responsible in generating In- ions.


The negatively charged polyiodideions can be pentaiodide I5-, triiodide I3-, or Heptaiodide [I7-]. These polyiodideions are charge donors and form complexes with Amylose. Brown color is the benchtop Lugol’s solution of iodine. The charge transfer complex of polyiodide and Amylose electrons absorb light energy, and are excited to higher energy levels. Human eyes perceive the complementary color as a blue-black colour.

The color of benchtop iodine is therefore brown. The colorless polyiodides I3-,I5-,I7- are the I3-,I5-, and I7-. The amylose-iodide compound is blue-black.


It is used to identify starch in samples. An IKI indicator can confirm the presence of starch.

All iodometric starch titrations that use the starch indicator are based on the principle of the iodine-starch test.


The intensity of the blue color decreases when the temperature is increased and the presence water-miscible solutions like ethanol, isopropyl alcohol, etc. are present. The reason is that temperature increases cause the amylose/iodine complex to dissociate. The temperature drops, which is why the helical structure in the amylose iodine complex becomes more stable, leading to the regeneration of the blue-black colour complex.

What is Starch?

Starch is simply one of the most important complex carbohydrate compound. It is both a polysaccharide- and glucoside resource for plants. It is renewable and biodegradable, making it a great raw material.

Starch molecules are made up of a large number glucose units, which are linked by glycosidic bond and are produced by vegetables and other plants through photosynthesis. Starch molecules are essential for plant cell growth, development and reproduction. Starch is extracted from plant products such as wheat, barley, maize and potatoes.

Why do we perform Test for Starch?

The iodine starch test is used primarily to check for the presence of carbohydrate. There are many types of carbohydrates in the food we eat, including starch and sugars.


Q. Iodine test is used to detect which of the following

A) Protein
B) Fat
C) Carbohydrate
D) Chitin

Ans: Carbohydrate

Q. what is the function of ethanol in iodine test?

When a leaf is boiled in alcohol like ethanol, it helps to extract chlorophyll and help remove the green color of the leaves. After the removal of chlorophyll, the color of the leaf changes to white, and it is easy to observe the change in the color of iodine as starch gives a blue color to iodine.

Q. why do we perform the iodine test?

The iodine test is used to check the availability of starch like carbohydrates in a sample. Starch changes to a deep “blue-black” colour upon addition of aqueous solutions of the triiodide anion, because of the production of an intermolecular charge transfer component (complex).

Q. iodine test is used to detect which disease in rice?

Rice leaves infected by tungro virus was diagnosed by treating with iodine-potassium iodide solution in the field and iodine-potassium iodide-lactic acid solution in the laboratory. By this technique tungro infected plants could be differentiated from similar symptoms due to other causes.

Q. in iodine test starch converts into which colour?

In iodine test starch converts into blue-black or purple.



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