2.9 Separation of Photosynthetic Pigments by Chromatography (Practical 4)

Summary

This video provides a detailed protocol for separating leaf pigments using chromatography, specifically with spinach leaves. It outlines the process from preparing the leaf extract by grinding it with acetone and sand to applying the pigment on chromatography paper. Viewers learn how to set up the chromatography process, monitor the solvent front, and measure the distances traveled by both the solvent and pigments. The video also explains how to calculate RF values to identify different pigments based on their movement, offering a practical approach to understanding plant biology.

Takeaways

• 🌿 Obtain spinach leaves or another plant to extract pigments.
• 🔬 Break the leaves into small pieces and grind them with sand and acetone in a mortar.
• 🧪 Aim for a concentrated pigment liquid at the bottom of the mortar.
• ✏️ Use a pencil to draw a line 2 cm from the bottom of chromatography paper.
• 💧 Apply a concentrated pigment spot on the pencil line using a pipette or capillary tube.
• 🌊 Prepare the running solvent, ensuring it doesn't exceed the pencil line on the paper.
• 👀 Place the chromatography paper in the solvent and monitor the solvent front as it moves up.
• 📏 Mark the endpoint of the solvent front and measure the distances traveled by both the solvent and pigments.
• 📊 Calculate RF values for each pigment by dividing the distance moved by the pigment by that of the solvent front.
• 🔍 Compare RF values against known published values to identify the pigments present.

Q & A

What is the main purpose of the protocol described in the video?

The protocol aims to separate leaf pigments using chromatography, specifically focusing on extracting pigments from spinach leaves.

What materials are needed to start the chromatography process?

You need spinach leaves, acetone, sand, chromatography paper, a pencil, and a running solvent, which is commonly a mixture of petroleum ether and acetone.

Why is it important to use a pencil rather than a pen on chromatography paper?

Pencil does not contain pigments that could interfere with the separation process, whereas ink from a pen may contain various pigments that can contaminate results.

How should the pigment be prepared before applying it to the chromatography paper?

The leaves should be ground in a mortar with sand and acetone until a concentrated pigment solution is obtained at the bottom of the mortar.

What role does the running solvent play in chromatography?

The running solvent carries the pigments up the chromatography paper, allowing them to separate based on their solubility and affinity for the paper.

What should you do once the solvent front has stopped moving?

You should mark the final position of the solvent front with a pencil and measure the distance it traveled from the original pencil line.

How do you calculate the RF value for each pigment?

The RF value is calculated by dividing the distance traveled by the pigment by the distance traveled by the solvent front, both measured in millimeters.

What does an RF value indicate in chromatography?

The RF value helps in identifying the pigments by comparing them to known published RF values for specific pigments.

What is a common RF range for carotene as mentioned in the video?

The RF value for carotene typically ranges from 0.89 to 0.98.

Where can one find additional resources or written protocols for this practical?

Additional resources, including written protocols, can be found on various educational websites, which provide support for conducting the chromatography experiment.