To remove chlorophyll from a leaf, you boil the leaf in water first, then transfer it to hot ethanol (alcohol), which dissolves the green pigment and turns the leaf white or pale yellow. The whole process takes about 10 to 15 minutes and is most commonly done as preparation for the iodine starch test in biology classes. Here’s exactly how it works and why each step matters.
What You Need
- A fresh leaf (from a plant that has been in sunlight for several hours)
- Ethanol or methylated spirits (70% or higher concentration works well)
- A beaker of boiling water
- A smaller beaker, test tube, or heat-safe container for the ethanol
- Forceps or tweezers
- A white tile or petri dish for examining the leaf afterward
Step-by-Step Extraction
Start by boiling water in a beaker on a hot plate or stove. Using forceps, submerge the leaf in the boiling water for about two minutes. This step kills the leaf cells and softens the cell walls, which makes it much easier for the alcohol to penetrate the tissue and reach the chlorophyll inside. You’ll notice the leaf becomes limp and may turn a slightly darker green.
Next, turn off the heat source. Place ethanol in a smaller container (a test tube or small beaker works well) and set that container into the hot water, creating a water bath. Transfer the boiled leaf into the ethanol. The ethanol will gradually heat up from the surrounding water and begin to dissolve the chlorophyll. Over the next five to ten minutes, you’ll see the alcohol turn green as it pulls pigment out of the leaf. The leaf itself will fade to a whitish or pale yellow color.
Once the leaf has lost its green color, remove it with forceps and briefly dip it back into the warm water. This rinse softens the leaf again, since ethanol makes it brittle and stiff. The leaf is now ready for whatever test you need to perform.
Why Ethanol Works
Chlorophyll is a magnesium-containing compound with a long hydrocarbon tail. That structure makes it completely insoluble in water, which is why boiling the leaf in water alone won’t strip the color. Organic solvents like ethanol, acetone, and ether dissolve chlorophyll readily because they can interact with that hydrocarbon tail in ways water cannot.
Ethanol is the go-to choice for most classroom and field settings because it’s effective, relatively safe compared to acetone or ether, and widely available. Research comparing common solvents has found that ethanol generally extracts more chlorophyll than acetone and produces the most consistent results across different types of plant material. Acetone is preferred in some professional laboratory protocols (environmental water testing, for instance, typically uses a 90% acetone solution), but for leaf decolorization, ethanol is simpler and works just as well.
Why You Must Use a Water Bath
This is the single most important safety point. Ethanol has a flash point of 14°C (57°F), meaning it releases flammable vapors at room temperature and will ignite instantly near an open flame or spark. Never heat ethanol directly over a Bunsen burner, candle, or gas stove.
The water bath method keeps the ethanol’s temperature controlled. Surrounding hot water heats the alcohol gently and evenly without ever exposing it to a direct flame. If you’re using an electric hot plate, turn it off before placing the ethanol container in the water. The residual heat is enough. If your setup requires a Bunsen burner to boil the water initially, extinguish the flame completely before introducing any ethanol to the workspace.
How to Tell Extraction Is Complete
A successfully decolorized leaf turns whitish, cream, or pale yellow. There should be no visible green remaining. If green patches persist, return the leaf to fresh ethanol in the water bath for a few more minutes. Thicker or waxier leaves take longer because the solvent has to work through tougher outer layers and denser cell structures. Thin leaves from common garden plants or deciduous trees typically clear in five minutes or less.
The ethanol in your container will be visibly green, sometimes deeply so. That color is the extracted chlorophyll. If you’re processing multiple leaves, the ethanol becomes saturated and loses effectiveness, so replace it with fresh solvent between batches.
Adjustments for Tough or Thick Leaves
Leaves with thick cuticles (the waxy outer coating), like those from evergreen shrubs or tropical plants, resist ethanol penetration. A few adjustments help. First, extend the initial boiling time in water to three or four minutes to more thoroughly break down cell walls. Second, you can lightly score the surface of the leaf with a razor blade or pin before extraction, giving the solvent direct access to interior cells. Third, using warmer ethanol (keeping the water bath at a rolling temperature rather than letting it cool) speeds things up considerably.
Very thick or succulent leaves may never fully decolorize with a simple water bath setup. For these, longer soaking in ethanol at room temperature (overnight, in a sealed container) can accomplish what a short hot extraction cannot. This cold extraction is slower but avoids the repeated heating that can make delicate tissues fall apart.
Using the Cleared Leaf for a Starch Test
The most common reason for removing chlorophyll is the iodine starch test. Chlorophyll’s deep green color masks the color change that iodine produces when it reacts with starch, so you need a white or near-white leaf to see results clearly.
After rinsing the decolorized leaf in warm water, spread it flat on a white tile and add a few drops of iodine solution (potassium iodide). Areas of the leaf that contain starch will turn a distinctive blue-black within a minute or two. Areas without starch remain the brownish-yellow color of the iodine itself. This test demonstrates which parts of the leaf were photosynthesizing, since photosynthesis produces sugars that the plant stores as starch. A leaf kept in darkness for 24 to 48 hours before testing (called “destarching”) will show no blue-black color, confirming that light is necessary for starch production.
Other Uses for Chlorophyll Removal
Beyond starch testing, cleared leaves are used in leaf skeleton art, botanical pressing, and microscopy. Artists and crafters sometimes soak leaves in a dilute solution of sodium hydroxide (washing soda) rather than ethanol, which removes both pigment and soft tissue to leave behind the vein network. For scientific microscopy, chlorophyll removal allows staining with dyes that highlight specific cell structures without the green pigment overwhelming everything under the lens.
If your goal is to collect the chlorophyll itself rather than discard it, simply save the green ethanol solution. Evaporating off the alcohol in a well-ventilated area leaves behind a concentrated chlorophyll residue that can be used for pigment chromatography experiments or absorption spectrum demonstrations.