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Separation of Pigments of Leaves and Flowers using Chromatography Technique

Last updated date: 22nd Mar 2024
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The Chromatography Technique

In this article the chromatography technique can be widely used to separate, purify, and also identify the compounds. The substance balances in the chromatography between a stationary phase and a mobile phase. Following are the different types of chromatography techniques:

  • In paper chromatography, paper soaked in liquid and a liquid are used as filters in the mobile and stationary phase to separate the components of the mixture. After the separation, the other components leave a spot on the paper.

  • In the thin layer chromatography technique, glass or metal is coated with a thin layer of aluminium or silica based on the availability and this sheet is used as a filter to separate the components of the mixture.

  • The column chromatography technique is similar to the layer chromatography technique but the only difference is the time taken for the process. 


The aim is to separate the pigments that are present in flowers and leaves by the paper chromatography technique and determine their Rf values.


Paper chromatography is mainly partition chromatography. In this paper chromatography, the stationary phase is defined as paper. The paper contains 22 per cent of water molecules absorbed on about 78 per cent of cellulose.

The components mixture separation takes place by partitioning the components between the mobile phase and stationary phase. The mobile phase travels via paper by the capillary action. Depending on the ways the solvent travels on the paper, there exist three types of chromatography, given as follows.

  • Ascending Paper Chromatography

  • Descending Paper Chromatography

  • Circular Paper Chromatography

The distribution takes place in a definite ratio that represents the solution's characteristic distribution coefficient.

The Rf coefficient ration is given as follows:

Rf=distance travelled by solute, distance travelled by the solvent

Various substances possess various Rf values. Rf depends on several factors which are listed below.:

  • Nature of the solvent

  • nature of the substance

  • Presence of impurities

  • Temperature

  • quality of the filter paper

If any compound is coloured, it can be easily located on chromatographic paper. If the same substance is colourless, however, a reagent can be used to treat it, which produces a characteristic colour. The term developer is given to this reagent. And iodine is the most frequently used paper chromatography developer.

Materials Required

  • Extract of leaves and extract of flowers

  • Whatman's filter paper

  • Acetone/Methanol

  • Acetone/Chloroform

  • Glass jar

  • Rubber cork fixed with a hook in the centre

  • Distilled water

  • Test tubes

  • Petroleum ether

Apparatus Setup

(Image will be uploaded soon)


  • Take the Whatman filter paper and then draw a line using a pencil above 4cm from one end.

  • Then, grind the flowers and leaves in a motor and transfer the paste into the test tube.

  • In the crushed material, add methanol or acetone, shake well and filter the respective mixture.

  • The filtrate is collected in a test tube to perform experiments.

  • With the help of a capillary tube, add one drop of the filtrate on the filter paper and allow it to dry.

  • After that, hang the filter paper in a jar containing 20ml of chloroform and petroleum ether.

  • Keep the same jar till the mobile phase rises to 2/3rd of the length of the paper.

  • Then, remove the filter paper from the jar and mark the solvent front.

  • Outline the spots using a pencil and allow the filter paper to dry.

  • Now, measure the distance between the centre of different spots and the solvent front about the reference line as indicated.

  • Then, determine the pigment number in the flowers and leaves extract.

  • Finally, calculate the Rf value of different spots using the expression.

Observations and Inference

The noticed Observations and Inferences can be recorded in the respective fields of the table, which is given below.

S. No

Name of the extract

Colour of the spot

Distance travelled by spot from the original line

Distance travelled by solvent from the original line

Rf values





Results and Discussions

  • The Rf value of the components of the leaves is _____.

  • The Rf value of the components of the flowers is _____.


  • Always choose a fine capillary tube.

  • Do not allow the spots to spread while spotting the test solution available on the paper.

  • Do not disturb the jar once the experiment is arranged as long as the development of the chromatogram has been completed.

  • Use the capillary finely drawn to place the spot on the same paper.

  • Prior to developing the spots, allow the paper strip to become dry perfectly.

  • Handle the organic solvents carefully.

Finding the Number of Pigments in Leaves with the Help of Chromatography

If there exist more samples, it's spots are bigger on the TLC plate.

Like when we spill coffee on a white shirt , the more the coffee, the larger the spot! In general, on a shirt, the water carries the solute, which is considered a poor example.

Remember that a sample is applied in a tiny spot that goes to thin-layer chromatography (TLC) glass or near the bottom of the paper or the plastic plate. Then, the plate is placed carefully in the appropriate solvent and moves in an upward direction uniformly. Solutes, which are otherwise called pigment molecules, dissolve. After that, move upward at various rates with the solvent while the paper or the TLC plate adsorbent wants to "hold on" to the molecules. This is the main reason why the molecules having different polarities are separated and hence generate an Rf value.

FAQs on Separation of Pigments of Leaves and Flowers using Chromatography Technique

1. What is an iodine spot?

The spot present on the chromatography paper strip where the acetone extract of the leaf is to be loaded is referred to as the iodine spot.

2. What is chromatography?

Chromatography is one of the processes of mixture separation laboratory techniques. Different components of the mixture are passed through a filter at varying speeds. The process is initiated by dissolving the mixture in a liquid called the “mobile phase”. It is then carried to a second phase also known as the stationary phase. Different components that are present in the mixture travel at different speeds in this second phase, this causes them to separate from each other. The time taken to travel by different components of the mixture is called “retention time”. The rate at which the components get separated is dependent on the nature of the secondary and mobile phases. Then, the mixture is dissolved in a mobile phase fluid that carries it via a structure that holds another material, which is known as the stationary phase. This chromatography technique was first used to separate the pigments of a mixture in the 19th century. This works on the principle that the smallest molecules travel faster up the liquid or mixture and the largest molecules travel at a much slower speed. 

3. Describe the advantages of chromatography compared to other techniques.

Chromatography is a technique used to separate different components of a mixture by sending them through a filter. The larger molecules of the mixture travel at a slower speed and the smaller particles of the mixture travel at a faster rate. The entire process has two different stages namely the mobile phase and stationary phase. The time taken to pass through the phase for a component is called the retention period and this technique was used for the very first time in the 19th century. Chromatography can be used in a wide variety of applications. This process is primarily used to separate complex mixtures. It works on various samples, including food particles, drugs, plastics, samples of air and water, pesticides, and extracts of tissue. Following are the advantages of using chromatography over the other methods:

  • Other separation techniques are not so accurate, but a precise separation of different components of the mixture can be done with the chromatography technique.

  • Unlike the other methods, chromatography needs very small and minute samples. 

  • Any samples ranging from drugs to tissue extracts can be processed through chromatography making it possible to use for diverse materials. 

It is very functional and efficient even in the case of complex materials. 

4. List some chromatographic techniques.

Chromatographic is a technique used to separate different components of the mixture by passing them through a filter at different speeds. It was used for the very first time in the 19th century. Following are the types and some techniques of chromatography: Column chromatography, paper chromatography, gas chromatography, and thin-layer chromatography.

  • In the paper chromatography technique, paper soaked in a liquid is used in the stationary phase and another liquid is used in a mobile phase as usual. Once the paper dries, these separated components appear as spots on the paper. 

  • Silica and aluminium are used as agents in the stationary phase and organic solvents are used in the mobile phase as filters in the liquid chromatography.

  • A plastic or glass layer coated with a thin layer of silica or aluminium is used as a filter in the thin layer chromatography technique and the separated components appear as small spots after the process.

  • Column chromatography is similar to the process of thin-layer chromatography but the only difference is the time taken to complete the process.

  • Any inert gas is used in the stationary phase and liquid is used in the mobile phase as filters to separate components of a mixture in gas chromatography and the separated materials are found with the help of a detector. 

5. Mention the factors that the Rf value of a compound depends on.

Rf means retention factor in chromatography. It is dependent on many factors like the layer thickness, moisture present on the TLC plate, temperature, depth of the mobile phase, sample size, and solvent parameters. The value of Rf is also dependent on the nature of the solvent, the nature of the compound, and the temperature.

  • If the component is bound to the absorbent more, it takes time to travel up the TLC plate and vice versa. The non-polar compounds move up faster whereas the polar compounds take time to move up the TLC plate. 

  • The component which moves up the TLC plate faster has a high Rf (retention factor) and the component which moves slower has less Rf (retention factor).