Theory & Procedure, Paper Chromatography | Additional Study Material for NEET PDF Download

Objective

Our objective is to separate and study plant pigments by paper chromatography.

Theory

Before going into detail, let’s understand the role of pigments in plants.

Photosynthetic plants convert light energy from the sun to chemical food energy.  During photosynthesis, molecules referred to as pigments are used to capture light energy.  Pigments are chemical compounds which reflect only certain wavelengths of visible light.  Plant leaves contain four primary pigments: chlorophyll a (dark green), chlorophyll b (yellowish-green), xanthophylls (yellow) and carotenoids (orange). 

To separate and visualize the four primary pigments of green plants, we can use a simple technique called chromatography.

What is Chromatography?

Chromatography is a technique used to separate molecules on the basis of differences in size, shape, mass, charge, solubility and adsorption properties. The term chromatography is derived from Greek words Chroma-colour and Graphe-write. There are many types of chromatography: paper chromatography, column chromatography, thin layer chromatography and partition chromatography. These techniques involve the interaction between three components: the mixture to be separated, a solid phase and a solvent.

How does paper chromatography work?

In paper chromatography, the mixture is spotted onto the paper, dried and the solvent is allowed to flow along the sheet by capillary attraction.  As the solvent slowly moves through the paper, the different compounds of the mixture separate into different coloured spots. The paper is dried and the position of different compounds is visualized. The principle behind the paper chromatography is that the most soluble substances move further on the filter paper than the least soluble substances. Different plant pigments can be separated by using the technique of paper chromatography.

What is Retention Factor or Rf value?

Retention factor or R_f value is applied in chromatography to make the technique more scientific than a mere analysis. The retention factor or Rf is defined as the distance travelled by the compound divided by the distance traveled by the solvent.
R_f=(Distance travelled by the compound)/(Distance travelled by the solvent)

Diagrammatic example that demonstrates Rf value:

Learning Outcomes

• Students will understand the principle behind chromatography techniques.
• Students will learn about different types of pigments occurring in a plant leaves.
• Students will learn how to calculate the Retention factor.
• Students will be able to do the experiment more accurately in the real lab once they understand the steps through the animation and simulation.

Now, let’s separate the pigments from fresh spinach leaves using paper chromatography. 

Materials required

Real Lab Procedure

• Take a few freshly plucked green spinach leaves.
• Using scissors,  cut the spinach leaves into small pieces and let them fall into the mortar.
• Take a measuring cylinder that contains 5ml of acetone and pour it into the mortar.
• Grind the spinach leaves using the mortar and pestle.
• Place the extract into a watch glass using a spatula.
• Take a strip of filter paper having a narrow notch at one end of the strip.
• Take a pencil and a scale and draw a horizontal line with a pencil about 2-3 cm away from the tip of the notch.
• Put a drop of the pigment extract in the middle of the line with the help of a capillary tube.
• Allow the drop to dry and repeat till four or five drops are placed on the paper.
• Take the chromatographic chamber and pour ether acetone solvent in it.
• Fold one end of the filter paper strip and staple it.
• Using a thread, hang the filter paper strip in the chromatographic chamber.
• The loading spot should remain about 1 cm above the solvent level.
• Leave the chromatographic chamber undisturbed for some time.
• We can observe, as the solvent moves through the paper, it spreads the different pigments of the mixture to various distances.
• When the solvent rises about 3/4th up the strip, remove the strip carefully and let it dry.

Simulator Procedure (as performed through Online Labs)

• You can select the type of leaf extract using the drop down list, ‘Select the plant pigment extract’.
• Now, select the type of solvent using ‘Select the solvent’ drop down list.
• Click on the ‘start’ button to start the experiment.
• We can observe that the different pigments move through the filter paper to various distances.
• Click on the test tube to measure the R_f value. There appears a tooltip to identify the band.
• Select the ‘show scale’ check box to measure the distance travelled by the pigment and solvent.
• Select the ‘show chart’ check box which displays a chart of predefined values of different leaf pigments.
• Check the calculated Rf value that matches the predefined values in the chart.
• Now select the appropriate pigment band from ‘Select the pigment identified’ drop down list.
• To repeat the experiment, click on the ‘Reset’ button.

Observation

The dried chromatographic paper strip shows four distinct paper bands. Different pigments can be identified by their colours.

Calculations

R_f Value of the each pigment spot can be calculated by the equation;

R_f=(Distance travelled by the compound)/(Distance travelled by the solvent)

Measure the distance of each pigment band from the loading spot and also the distance travelled by the solvent. Calculate the Rf value using the equation and record the values in the table.

Result

The topmost orange yellow band of pigments in the separation corresponds to carotene. The yellowish band appearing below it indicates the xanthophylls. The third from above dark green band represents chlorophyll a. The lowermost yellowish green band is that of chlorophyll b.

Precaution

• Spinach leaves should be fresh and green.
• The loading spot should be 2-3 cm away from the tip of the notch.
• While hanging the strips in the chromatography chamber, the loading spot should remain about 1 cm above the solvent level.