Separation Of Plant Pigments Through Paper Chromatography

A plant contains several pigments that enable its metabolic capabilities. Chlorophyll, of course, is the most important plant pigment in the leaves of photosynthetic plants. It is what gives most plants their characteristic green colour and mediates the processes of photosynthesis as well. Besides chlorophyll, plants contain a group of pigments called carotenoids, yellow-orange pigments. Xanthophyll and carotene are the two most important carotenoids. How does one separate these pigments of a plant from visualising their colour? Carry out this simple yet interesting experiment to find out! 

Table of Content

• Aim

• Apparatus required

• Theory

• Procedure

• Observations

• Result

• Precautions

Aim

To separate and visualise plant pigments present in spinach leaf using the technique of paper chromatography

Apparatus Required

Spinach leaves, pestle and mortar, water, chromatography jar, ether acetone solvent, scissors, Whatman No.1 filter paper strips, watch glass, capillary tube, pencil, scale

Theory

• A typical photosynthetic plant contains several pigments across its stems, leaves and flowers. 

• The leaves of a photosynthetic plant contain the photosynthetic pigments chlorophyll a and chlorophyll b, which impart green colour to the plant, and are contained in the chloroplasts. 

• They form the reaction centres of photosynthesis by absorbing the incident sunlight. 

• The second most commonly occurring plant pigments are the carotenoids- namely, xanthophyll and carotene. The carotenoids are the yellow-orange pigments of the plant, present within chromoplasts. 

• The different pigments occurring in the leaves of a plant can be visualised using paper chromatography. 

Principles of Paper Chromatography

• Martin and Synge developed paper chromatography in the year 1943. It is a simple technique used to visualise different components within a mixture.

• Paper chromatography is based on the different solubilities of the various components of a mixture in a particular solvent. Based on their solubilities in the solvent, each component is separated. 

• Paper chromatography comprises two phases- the stationary phase, which is a nonpolar medium of cellulose and water, and the mobile phase, which is a polar, organic solvent (e.g. ether acetone solvent) 

• The mixture is loaded onto the paper, which is then suspended in an appropriate solvent (the mobile phase)

• Absorption, solubility and capillarity are the three factors that affect the separation of the components and the distance travelled by each component from the initial spot. 

• Absorption holds the substance onto the paper. Capillary action pulls up the substances through the paper. Different solubilities separate the components in the mixture.

• The most soluble and lighter substance moves farthest, while the least soluble and heavier substance separates early on near the loading point on the paper

• The separated pigments appear as streaks at different positions on the chromatography paper. 

Procedure

1. Take a few fresh spinach leaves and wash them under distilled water 

2. Using your scissors, cut the spinach leaves into small pieces and add them into the mortar, along with a few drops of acetone (~5mL). Grind into a fine paste

3. Take a strip of Whatman filter paper No.1 (6” x 1.5”) and cut one end such that it has a tapering (arrowed) notch 

4. Using a pencil, mark a horizontal line 2 cm above the tip of the notch 

5. Add just one drop of the prepared leaf paste onto the centre of this horizontal line using a capillary tube 

6. Once the drop dries, add another drop of the sample onto the same spot 

7. Repeat steps 5-6 at least 4-5 times. This will ensure a concentrated sample. 

8. Add ~5mL of the ether acetone solvent into the chromatography jar

9. Now suspend the loaded filter paper onto the hook of the cork of the chromatography jar. For this step, one can also use a measuring cylinder, a cork, and a pushpin. 

10. The pointed end of the paper should just touch the solvent once suspended. The horizontal line marked on the paper MUST NOT TOUCH the solvent 

11. Allow the solvent to rise through the filter paper. This may take up to 40 minutes.

12. Once the pigments have separated into four distinct bands, and the solvent has stopped rising, remove the paper from the hook. 

13. Mark the edge of the solvent on the paper. This is the solvent front. 

14.  Allow the paper to dry completely. 

Observations 

The chromatogram displays four distinct bands of colour, indicating the separation of the leaf pigments. 

Result

The pigments within the mixture have separated into four distinct bands as follows, from the bottom up to the solvent front:

• Band of green colour- chlorophyll b

• Band of blue-green colour- chlorophyll a

• Band of yellow colour- xanthophyll

• Band of orange colour- carotene

Separation of leaf pigments using paper chromatography

Precautions

1. Do not use a ball or ink pen to mark the chromatography paper.

2. Ensure that the loaded spot is not touching the solvent and is visible above it

3. Make sure to transfer a generous amount of the sample onto the paper to ensure better results 

4. Be careful not to handle ether acetone near any flame source, as this solvent is highly flammable. 

Lab Manual Questions

1. Which photosynthetic pigment travels the farthest and why?

Ans: Carotene travels the farthest, near the solvent front. This is because it is more soluble in the solvent than the other three pigments and doesn’t bond to the medium of the paper via hydrogen bonds. 

2. Why is acetone used as a solvent instead of water? 

Ans: Acetone is less polar than water. This allows for greater allows greater resolution, i.e., better separation of the pigments in the mixture 

3. What will happen if the spot touches the solvent?

Ans: It is important to ensure that the sample doesn’t touch the mobile phase. If it does, the mixture spot-loaded will dissolve immediately in the solvent, thereby maligning the entire experiment

Viva Questions

1. How many pigments have you separated from the sample? What are they?

Ans: Four pigments have been separated from the sample, chlorophyll a, chlorophyll b, xanthophyll and carotene 

2. Why do you think acetone is used as the mobile phase in this experiment, not water?

Ans: Ether-acetone is a non-polar solvent. The natural pigments present in the leaves are, however, polar. This is important since separation in paper chromatography is based on the differing solubilities of the pigments in the solvent and the stationary phase. 

3. Based on your experiment, what can you infer about the polarities of the separated pigments?

Ans: The most polar pigment is chlorophyll b, followed by chlorophyll a, xanthophyll, and finally, carotene, which is the least polar, hence most soluble in the non-polar solvent.

4. What do you know about the polarity of the chromatography paper?

Ans: The chromatography paper is made up of cellulose, attracting water vapour molecules, thereby contributing to the polar nature of the paper. 

5. How many different types of pigments occur in plants? Can you name these pigments?

Ans: Four types of pigments occur in plants- the chlorophylls, and the carotenoids, which include carotene and xanthophylls like lutein, anthocyanin, and betalains. 

6. We are well aware that chlorophylls are involved in photosynthesis. What are the functions of carotenoids?

Ans: Carotenoids are also involved in photosynthesis. They absorb wavelengths not received by chlorophylls and transfer the absorbed energy to the chlorophyll molecules. Hence, they are also called accessory pigments. They are also involved in photoprotection. 

7. Apart from paper chromatography, what other types of chromatographies are used for separation purposes?

Ans: Thin layer chromatography, column chromatography, gas chromatography, ion-exchange chromatography, affinity chromatography, etc., are the other types used to separate components in a mixture. 

8. What are the absorption spectrum values of chlorophyll a and b?

Ans: Chlorophyll a absorbs the maximum wavelength of 642 nm and 372nm (red and blue regions, respectively), while chlorophyll b absorbs a maximum wavelength of 626 nm and 372 nm (red and blue regions, respectively). 

Practical Based Questions 

1. The pigment that appears blue-green on the chromatogram is?

1. Chlorophyll a

2. Chlorophyll b

3. Xanthophyll

4. Carotene 

Ans. A. Chlorophyll a

2. The pigment that is most soluble in the solvent is:

1. Chlorophyll a

2. Chlorophyll b

3. Xanthophyll

4. Carotene 

Ans. D. Carotene 

3. The stationary phase employed in the paper chromatography experiment is:

1. Acetone

2. Ether-acetone 

3. Water

4. Paper

Ans. D. Paper

4. Which of the following is not involved in separating leaf pigments in paper chromatography?

1. Adsorption

2. Solubility 

3. Capillarity 

4. Gravitation

Ans. D. Gravitation 

5. Xanthophyll belongs to _____ group of plant pigments

1. Chlorophylls

2. Porphyrins

3. Carotenoids

4. Flavonoids

Ans. C. Carotenoids

6. The P700 reaction centre comprises 

1. More of chlorophyll a than chlorophyll b

2. More of chlorophyll b than chlorophyll a

3. Equal amounts of chlorophyll a

4. Only chlorophyll a 

Ans. A. More of chlorophyll a than chlorophyll b

7. The pigment that is least soluble in the mobile phase is 

1. Chlorophyll a

2. Chlorophyll b

3. Carotene 

4. Xanthophyll

Ans. B. Chlorophyll b 

8. The pigment that travels farthest on the paper is 

1. Most soluble in the solvent

2. Least soluble in the solvent 

3. Not soluble in the solvent

4. Of lower molecular weight than the other pigments 

Ans. A. Most soluble in the solvent

Conclusion

• A leaf from a typical photosynthetic plant comprises four pigments: chlorophyll a and b, xanthophyll and carotene. 

• These pigments can be separated using the paper chromatography technique, using acetone as the mobile phase/ solvent. 

• Four bands of separated pigments are observed, with chlorophyll b at the bottom, followed by chlorophyll a, xanthophyll and finally, carotene at the top.