Which one is the most suitable way to separate Chlorophyll from dried leaf extract?

This easy science project shows simple and efficient method of chlorophyll extraction from a plant. Attention! This method involves usage of flammable, volatileand toxic substances. All work with solvents should be conducted on the open air or in actively ventilated room. Adult supervision recommended for this project! Required Materials (in order of appearance): Note: solvent mixtures can give better results in extraction of the full complex of plant pigment then individual solvent. You could study how good are the different solvents and their mixtures for chlorophyll extraction. (Good chemistry science fair project idea, by the way). Extraction Procedure. But this is a different story.

This is only a demonstration of working method which can be refined and used for a full scale science project.

Plants contain many pigments in different proportions. For flowering plants it's mostly a combination of chlorophyll-A, chlorophyll-B, pheophytin and Carotenes. This pigments have shades of green, blue green, grayish, and yellow-orange. Their concentration is different for different species and also depend on the time of year. Also, this pigments have different solubility in different solvents.

Extracted pigments could be used in another projects, such aschromatography.

Dark green leaves of any plant.
Cup.
Scissors.
Water.
Paper towel.
Foil.
Oven.
Mortar and pestle.
Glass test tubes with lids or small glass jars with tight lids.
Tea or coffee. (optional)

You may try one of the following solvents:

Rubbing alcohol/ethanol
Isopropanol
Acetone
Hexan

All this solvents can be used for chlorophyll extraction. However they have different property called polarity. This property affects their efficiency as chlorophyll solvent.

For our tests we used isopropanol and hexan/benzin mixture.

Another note: as a general rule solvents must be as chemically clean as possible. For example nail polish may not work. Even though it mostly contains acetone it also contains other chemical compounds which may react with your sample.

Put leaves in the cup, pour in boiling water until it covers the sample. Leave for ~30 sec – 1 min. Remove leaves from water and dry them with paper towel.

Boiling water kills the cells and destroys enzymes which can promote chlorophyll degradation. It also breaks chloroplatsts which makes chlorophyll extraction easier.

Take the leaves and remove petioles and central veins – this parts of leaf do not contain a lot of pigments. Throw them away. Cut the rest of the leaf material into the small 1-2 mm pieces. Spread them evenly on the foil and put it in the oven for 20 minutes at temperature 104F (40C). Have a cup of tea.

Put dry leaf pieces in the mortar (make sure mortar and pestle are perfectly dry!) and grind them. Continue until they turn into uniform yellow-green powder.

Grinding breaks cell walls and at the same time increases area of the surface. Leaf grinds will release more pigment.

Put powder in a test tube with tight lid. Add few ml of solvent, close the lid and shake

Note: try to use reasonably small amount of solvent. Five to ten ml is a good volume. Solvents are toxic and the less you deal with them the better. Make sure that your working place is well ventilated!

Have a cup of coffee.After coffee break check your chlorophyll. There should be emerald-green slightly opalescent liquid on top of dark green powder (which will sink to the bottom of the test tube)This is it. Chlorophyll is extracted. At the moment it's pretty diluted and solution contaminated with fine debris from the broken cells. Remains of chloroplasts and other cell components are floating in liquid making it slightly foggy. To wrap this experiment nicely we would recommend clean and concentrate extracted pigments.

Since we don't have centrifuge let's make time and gravity work. In other, more scientific words we'll do sedimentation of the leaf cells debris (two more jars/test tubes needed).

Here is how:

Wait for another 10-15 minutes for the leaf powder to settle on the bottom of the test tube.

Extremely carefully pour 90% of emerald-green liquid into second test tube. Close with tight lid. Put it in a cool dark place for few hours (24 is a good number).

Very carefully pour 90% of the liquid from the second test tube to the third test tube. This time leave it open and place it in well ventilated place. Check it from time to time. All used solvents are pretty volatile. After a while most of solvent is gone and what left is a pretty clean and concentrated plant pigments extract.

To check the composition of the extracted pigments put drop of the extract on the chromatography plate....

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