Experiment Report: Chromatography of Plant Pigments

Abstract

The experiment aimed to separate and analyze plant pigments using paper chromatography. The photosynthetic pigments in spinach leaves, chlorophyll b, and carotenoid, were identified, and their separation behavior was observed. The principles of capillarity and solubility were crucial in conducting the experiment.

Introduction

Photosynthesis, a fundamental process in plants, requires adequate light. Pigments, such as chlorophylls and carotenoids, are responsible for absorbing visible light. Each pigment has a specific absorption spectrum, indicating its ability to absorb light at different wavelengths.

For chlorophyll a, blue and red light are most effective for photosynthesis.

There is a strong correlation between the absorption spectra of leaves and the absorption spectra of chlorophyll a, chlorophyll b, and carotenoids. These pigments play a vital role in absorbing visible light for photosynthesis in leaves.

Experimental Technique

In this experiment, we employed paper chromatography to separate and analyze plant pigments. Capillarity, the phenomenon of liquid rising in narrow spaces, played a significant role in the separation process.

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Although chlorophyll b has a higher molecular mass than chlorophyll a, it exhibits greater capillary rise due to its lighter nature. This sensitivity allows for the differentiation of pigments based on their molecular structures.

Chromatographic Strip and Pigments

Our experimental results revealed that carotenoid has the lightest molecular weight. Its ratio of front is lower than that of chlorophyll b, indicating that it can cause more rise between the microscopic spaces between the fibers of the chromatographic strip. There is a direct correlation between molecular weight and the separation behavior of pigments; as the molecular weight decreases, the respective colored band for each pigment rises higher.

Physical Phenomena

Two physical phenomena make paper chromatography possible: capillarity and solubility. Capillarity allows the solvent to move through the chromatographic strip, while solubility determines how well the pigments dissolve in the solvent.

Other Applications

Paper chromatography has various applications beyond the separation of plant pigments. It is also used for separating amino acids and sugars.

Materials and Methods

Materials:

• Chromatography paper strips
• Capillary tubes
• Solvent mixture
• Spinach leaves (source of pigments)

Methods:

1. Handle chromatography paper strips carefully, touching only the edges and one end to avoid errors during capillary action.
2. Use a capillary tube to spot the pigment on the paper strip, ensuring only a small spot is applied and respotting until a dark green spot is produced for reliable results.
3. When placing the strip in the solvent, ensure the pigments' extract spot is not immersed in the solvent.
4. The chromatogram is lowered into the solvent, allowing pigments to migrate.
5. Stop the process when the solvent front nears the top of the paper.

Results

Table 1: Pigment Separation

Discussion

The experiment successfully demonstrated the separation of plant pigments using paper chromatography. It highlighted the significance of capillarity and solubility in the process. We observed that carotenoid had the lightest molecular weight, which affected its migration behavior on the chromatographic strip.

Furthermore, the experiment emphasized the practical applications of paper chromatography, including the separation of amino acids and sugars.

Limitations

As this is a qualitative experiment, several precautions were necessary:

• Handle chromatography paper strips carefully, touching only the edges and one end to avoid errors during capillary action.
• Use a capillary tube to spot the pigment on the paper strip, ensuring only a small spot is applied and respotting until a dark green spot is produced for reliable results.
• When placing the strip in the solvent, ensure the pigments' extract spot is not immersed in the solvent.
• Time constraints during the experiment could affect precision. Allocating more time for careful execution is advisable, especially during the respotting of pigment spots, ensuring each spot is dry before applying another.

Conclusion

Chromatography is a valuable technique for differentiating various pigments in plants. In the case of photosynthetic pigments in spinach, we identified chlorophyll b and carotenoid. Theoretical expectations suggest the presence of four pigments, including chlorophyll a and xanthophyll. During the chromatography process, a solvent mixture is selected to separate individual pigments. The loaded chromatogram is then immersed in the solvent, allowing pigments to migrate. The process is stopped when the solvent front nears the top of the paper.