Chlorophyll is effectively separated from other leaf pigments using paper chromatography, a powerful technique that leverages the differential movement of molecules based on their varying polarities and affinities for the stationary and mobile phases.
Understanding the Separation Principle
Paper chromatography separates the pigments in the leaf based on the distance travelled by pigment molecules on the paper in a nonpolar solvent. This separation relies on two key interactions:
- Stationary Phase: The chromatography paper, typically made of cellulose, acts as the stationary phase. Cellulose is a polar substance due to its many hydroxyl groups.
- Mobile Phase: A nonpolar solvent mixture (e.g., petroleum ether, acetone, or ethanol) acts as the mobile phase, moving up the paper by capillary action.
Pigments interact differently with these two phases. Less polar pigments will dissolve more readily in the nonpolar solvent (mobile phase) and travel further up the paper. More polar pigments will be more attracted to the polar paper (stationary phase) and will travel shorter distances.
The Process of Separating Chlorophyll and Other Pigments
To separate chlorophyll from a leaf extract using paper chromatography, follow these general steps:
1. Preparing the Pigment Extract
- Leaf Selection: Choose fresh, healthy leaves (e.g., spinach, kale).
- Grinding: Crush the leaves thoroughly using a mortar and pestle.
- Solvent Addition: Add a small amount of a suitable solvent (e.g., acetone or ethanol) to extract the pigments. This solvent dissolves the chlorophyll and other pigments from the leaf cells.
- Filtration: Filter or decant the liquid to obtain a clear, concentrated pigment solution.
2. Preparing the Chromatography Setup
- Chromatography Paper: Cut a strip of chromatography paper (or filter paper) to fit your container. Draw a pencil line about 1-2 cm from one end – this is the origin line.
- Applying the Extract: Carefully apply a small, concentrated spot of the pigment extract onto the center of the origin line using a capillary tube or a fine brush. Allow it to dry completely. Repeat this step several times to get a concentrated spot.
- Developing Solvent: Prepare a nonpolar solvent mixture (e.g., 9:1 petroleum ether to acetone, or similar ratios of other nonpolar solvents) in a suitable container (e.g., a tall beaker or jar). The solvent level should be below the origin line when the paper is placed inside.
3. Running the Chromatography
- Placement: Carefully suspend the paper strip in the container so that the bottom edge, where the pigment spot is, is just touching the solvent. Ensure the pigment spot itself is above the solvent level.
- Elution: Cover the container to saturate the atmosphere with solvent vapor, which helps maintain a consistent solvent front. As the solvent moves up the paper by capillary action, it dissolves the pigments and carries them along.
- Separation: Due to their differing solubilities and affinities, each pigment will travel at a different rate, separating into distinct bands on the paper.
4. Observing and Identifying Pigments
Once the solvent front reaches near the top of the paper (or a predetermined height), remove the paper and immediately mark the solvent front with a pencil. Allow the paper to dry. You will observe distinct colored bands:
- Carotenes: Travel furthest, appear yellowish-orange. These are the least polar.
- Xanthophylls: Travel next, appear yellow. Slightly more polar than carotenes.
- Chlorophyll a: Appears blue-green, travels further than chlorophyll b. Less polar than chlorophyll b.
- Chlorophyll b: Appears yellow-green, travels shorter distances. More polar than chlorophyll a.
The image below illustrates the typical separation:
Solvent Front
↑
| Carotenes (Yellow-Orange)
| Xanthophylls (Yellow)
| Chlorophyll a (Blue-Green)
| Chlorophyll b (Yellow-Green)
|
------------------ Origin Line (Initial Pigment Spot)
| Mobile Phase (Solvent)Key Pigment Characteristics and Relative Positions
The relative positions of the pigments are consistent due to their inherent polarities. Here's a quick overview:
| Pigment | Approximate Color | Polarity (Relative) | Distance Traveled (Relative) |
|---|---|---|---|
| Carotenes | Yellow-Orange | Least Polar | Furthest |
| Xanthophylls | Yellow | Low Polar | High |
| Chlorophyll a | Blue-Green | Moderate Polar | Medium-High |
| Chlorophyll b | Yellow-Green | Higher Polar | Medium-Low |
| Other Polar* | Various (e.g., Anthocyanins) | Most Polar | Shortest (or stay at origin) |
Note: Anthocyanins require a different, more polar solvent system for effective separation and are not typically seen with the nonpolar solvents used for chlorophylls/carotenoids.
By observing these distinct colored bands, chlorophyll a and chlorophyll b can be easily identified and separated from other pigments present in the leaf extract using paper chromatography.
