Paper chromatography separates the various pigments found in leaves by exploiting their different solubilities and affinities for the paper (stationary phase) and a solvent (mobile phase), allowing you to see the vibrant array of colors hidden within.
The Fundamental Principle of Chromatography
Chromatography is a laboratory technique used to separate mixtures. It relies on a stationary phase (the paper in this case) and a mobile phase (a solvent, like alcohol). Components of a mixture move at different speeds through the stationary phase because they have varying degrees of attraction to both the stationary and mobile phases. Those that are more soluble in the mobile phase and less attracted to the stationary phase will travel further up the paper.
Step-by-Step Process with Leaves
To perform paper chromatography with leaves, a few key steps are involved:
- Preparation of Leaf Extract: First, plant cells are broken open to release their pigment molecules. This is typically done by grinding up leaf pieces with a small amount of a solvent, commonly alcohol (such as rubbing alcohol or ethanol). This creates a concentrated solution of plant pigments.
- Setting Up the Chromatogram: A solution of plant matter and alcohol is placed at the bottom of a piece of paper. This spot should be above the level of the solvent that will be introduced later. Specialized chromatography paper, which is highly absorbent, is ideal for this purpose.
- Separation: The bottom edge of the paper, with the pigment spot, is then carefully placed into a small amount of the mobile phase solvent (e.g., alcohol or a solvent mixture) in a tall container. As the alcohol moves up the paper by capillary action, taking pigment molecules with it.
- Observation: As the solvent front moves up, it carries the pigments along. Different pigments travel at different rates, forming distinct colored bands or "streaks" up the paper, revealing colors like yellow, orange, and various shades of green.
Why Pigments Separate
The separation of pigments occurs due to differences in their:
- Solubility in the Mobile Phase: Pigments that are more soluble in the alcohol solvent will dissolve readily and be carried further up the paper.
- Adsorption to the Stationary Phase: Pigments that are more strongly attracted to (adsorbed by) the cellulose fibers of the paper will tend to move slower and not travel as far.
- Molecular Size and Polarity: Generally, smaller, less polar pigments tend to travel faster and further because they are more soluble in less polar solvents (like alcohol) and interact less with the polar paper.
The combination of these factors dictates how far each pigment travels, resulting in their separation into distinct bands. This allows scientists and students to calculate an "Rf value" (retardation factor) for each pigment, which is the ratio of the distance the pigment traveled to the distance the solvent traveled.
What You See: Leaf Pigments Revealed
A typical chromatogram of a green leaf will reveal several distinct pigment bands, each with a different color:
| Pigment Type | Color | Relative Movement (General) | Primary Role |
|---|---|---|---|
| Carotenoids | Yellow-Orange | Travels Farthest | Photosynthesis (accessory), Photoprotection |
| Xanthophylls | Yellow | Travels Farther | Photosynthesis (accessory), Photoprotection |
| Chlorophyll a | Blue-Green | Travels Moderately | Primary photosynthetic pigment |
| Chlorophyll b | Yellow-Green | Travels Least | Accessory photosynthetic pigment |
| Anthocyanins | Red-Purple | (May not be present or separate well, often water-soluble) | Photoprotection, typically in autumn |
Practical Applications and Insights
Paper chromatography with leaves is a widely used and insightful experiment for several reasons:
- Demonstrates Hidden Colors: It visually demonstrates that even seemingly plain green leaves contain a rich spectrum of pigments, particularly carotenoids and xanthophylls, which are often masked by the dominant chlorophylls during the growing season.
- Explains Autumn Colors: It helps explain why leaves change color in autumn: as chlorophyll breaks down, the underlying yellow and orange carotenoids become visible.
- Educational Tool: It's an excellent hands-on activity for teaching principles of chemistry, biology, and plant physiology in an accessible way.
- Research: While basic, chromatography is fundamental to more advanced separation techniques used in plant science research.
