Chlorophyll can be extracted by using organic solvents that vary in polarity.
Several factors affect the extraction process, and selecting the appropriate solvent is critical for a successful extraction. Here's a breakdown of how chlorophyll extraction works:
Selecting the Right Solvent
The choice of solvent is paramount. Common solvents used include:
- Acetone: Effective, but highly volatile and toxic. Anhydrous acetone is preferred to prevent chlorophyll degradation.
- Ethanol: A safer alternative, but may not be as effective as acetone for some plant materials.
- Methanol: Similar to ethanol in terms of safety, but extraction efficiency can vary.
- Petroleum Ether: Used in combination with other solvents for separating chlorophyll from other pigments.
- Diethyl Ether: Similar to petroleum ether in use.
The optimal solvent depends on the type of plant material and the desired purity of the chlorophyll extract.
Extraction Procedure: A Step-by-Step Guide
- Sample Preparation: Grind the plant material (e.g., leaves) into small pieces to increase the surface area for better solvent penetration.
- Solvent Addition: Add the chosen organic solvent to the ground plant material in a suitable container. The volume of solvent should be sufficient to completely submerge the plant material.
- Extraction:
- Maceration: Allow the mixture to sit for a specific period (e.g., 24 hours) at room temperature in a dark place. Stir or shake the mixture periodically to enhance extraction.
- Soxhlet Extraction (optional): For more efficient extraction, a Soxhlet extractor can be used. This continuously cycles the solvent through the sample.
- Sonication (optional): Using an ultrasonic bath can also help to break the plant cell walls and release chlorophyll.
- Filtration: Filter the mixture through filter paper or cheesecloth to remove solid plant debris. This step yields a crude chlorophyll extract.
- Separation (Optional): To separate chlorophylls a and b, techniques like thin-layer chromatography (TLC) or column chromatography can be used. These methods utilize the differential solubility of the two chlorophyll forms in various solvents.
- Evaporation: Evaporate the solvent using a rotary evaporator or under a stream of nitrogen gas. This concentrates the chlorophyll extract. Caution: Chlorophyll degrades easily when exposed to light and heat, so perform this step carefully.
Factors Affecting Chlorophyll Extraction
- Plant Material: The type of plant, its age, and its physiological state can influence the chlorophyll content and extractability.
- Solvent Polarity: The polarity of the solvent should be compatible with the polarity of chlorophyll.
- Temperature: High temperatures can degrade chlorophyll. Extraction is best performed at room temperature or lower.
- Light: Chlorophyll is light-sensitive. Extraction should be carried out in the dark or under subdued light.
- Time: The extraction time should be optimized to maximize chlorophyll yield without causing degradation.
Example Procedure
- Finely chop spinach leaves (about 10 grams).
- Grind the leaves with 20 mL of 80% acetone in a mortar and pestle.
- Filter the mixture through filter paper.
- Collect the filtrate (the green chlorophyll extract).
- Proceed with further purification or analysis as needed.
