Biochemical Test

Iodine Test Principle, Definition, Procedure, Result, Uses, Limitation

Iodine testing is a chemical test that distinguishes mono- or diaccharides from polysaccharides such as amylase, glycogen, and dextrin. Starch-iodine is a...

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Sourav Bio
This article writter by Sourav Bio on December 20, 2021

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Iodine Test Principle, Definition, Procedure, Result, Uses, Limitation
Iodine Test Principle, Definition, Procedure, Result, Uses, Limitation

Definition of Iodine Test

Iodine testing is a chemical test that distinguishes mono- or diaccharides from polysaccharides such as amylase, glycogen, and dextrin. Starch-iodine is a variant of this test. It’s used to determine if there is glucose in the leaves.

Objectives of Iodine Test

To detect the existence of polysaccharides, primarily starch.

Principle of Iodine Test

The iodine test relies on the fact that polyiodide ions form colored adsorption compounds with helical chains containing glucose residues of amylase, dextrin, or glycogen. Colorless remains monosaccharides and disaccharides as well as branched polysaccharides such cellulose. Amylopectin gives off an orange-yellow color.

Lugol’s Iodine is the reagent in the iodine testing. It is an aqueous solution containing elemental iodine as well as potassium iodide. Iodine is not soluble in water by itself. The addition of potassium iodine causes a reversible reaction between the iodine and iodine, which forms a triiodide. This reacts with an Iodine mole to create a pentaiodide. The color of the bench iodine solution is brown; however, the triiodide and pentaiodide solutions are all indistinguishable.

This test is based on the helix structure (coil or spring) of the glucose chains. The length of the glucose chain determines the color. The pentaiodide and triiodide ions that are formed are linear and slip within the helix structure. The complex’s color is due to the fact that the charge transfer between the helix ions and the polyiodide ions causes changes in the energy levels. This can absorb visible light which gives it its color.

With increasing temperature and presence of water-miscible compounds such as ethanol, the intensity of the color will decrease. The blue color amylase/iodine complex is destroyed by heating and reassembled on cooling. This is because the helical structure has been disrupted. Amylose then loses its iodine binding ability and the blue color. Cooling causes the blue color to return due to the recovery and regaining the helical structure.


  • 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. One ml of the given sample should be taken in a clean, dry tube.
  2. You can take control of 1 ml distilled water in a different tube.
  3. Mix the two tubes together with about 2-3 drops of Lugol’s solution in a bowl.
  4. You can observe the color appearance in the test tubes.
  5. Then heat the tubes in the hot water bath until they are completely dry.
  6. Cool the test tubes by taking them out
  7. Take note of the color appearance in the test tubes.
Procedure of Iodine Test
A diagrammatic flow chart of the Iodine test. 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

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

  • A starch test can be performed on a sample to detect it.
  • The iodine testing can be used to help distinguish starch from monosaccharides and disaccharides as well as other polysaccharides.
  • The iodine testing is used to distinguish between starch, glucose, and carbohydrate.
  • For the detection of hyper- or hypothyroidism, blood iodine testing can be used.
  • Starch hydrolysis also uses the principle of starch iodine testing.
  • The basis for iodometric starch titrations is the iodine testing.

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.

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