Leaf Pigment Chromatography

One of the most popular high school biology chromatography labs is separating plant pigments from leaves using paper chromatography. In this leaf pigment chromatography experiment, students extract pigments such as chlorophyll, carotene, and xanthophyll from leaves or spinach and observe how the pigments separate along chromatography paper.

This paper chromatography lab provides a visual and hands-on way for students to connect molecular properties to key biological concepts including photosynthesis, plant structure, polarity, and solubility. Because the pigments separate into distinct colored bands, students can easily observe how chromatography separates mixtures based on differences in molecular behavior.

Spinach leaves are commonly used in chromatography labs because their pigments separate clearly during paper chromatography. Depending on the time of year, students can also compare pigments from leaves collected from trees, shrubs, and other green plants.

Why Plant Pigments Are Perfect for Teaching Chromatography

Plant pigments provide an excellent example of how chromatography separates mixtures. When a leaf extract is placed on chromatography paper and exposed to a solvent, the pigments move at different rates depending on their polarity and solubility. This makes plant pigment chromatography one of the clearest classroom demonstrations of chromatography principles.

This paper chromatography lab is especially effective in high school biology classes because it reinforces both content knowledge and essential lab skills. As students analyze the separation of leaf pigments, they practice careful observation, data analysis, and experimental design while gaining a deeper understanding of how plants capture and use light energy. Leaf pigment chromatography is a low-prep, high-impact lab that fits naturally into units on photosynthesis, plant biology, and scientific investigation. 

Check out this blog post on Paper Chromatography for additional photos and information. If you are teaching plant biology or photosynthesis, you may also be interested in this classroom activity that helps students investigate how water moves through leaves. In this plant transpiration lab, students measure how environmental factors affect the rate of transpiration and connect those results to plant structure and function.

Why Are Spinach Leaves Used in Chromatography Labs?

Spinach leaves are commonly used in chromatography labs because they contain several easily visible pigments, including chlorophyll and carotenoids. These pigments separate clearly during paper chromatography, making spinach an excellent choice for classroom experiments involving photosynthesis and plant pigments.

Although spinach works especially well, students can also use leaves collected from trees, shrubs, and other green plants. Depending on the time of year, different groups of students can test different types of leaves and compare the chromatography results. This extension helps students observe how pigment composition varies among plants while reinforcing experimental design and data analysis skills.

Leaf Pigment Chromatography Experiment

Here are photos that I took as my students were working on their lab. In this chromatography experiment, students prepare pigment extracts, apply the pigments to chromatography paper, and observe how the pigments separate into distinct colored bands.

Spinach leaves are placed in a mortar and pestle along with a little acetone.  Students grind the leaves until the acetone turns a dark green.  A small piece of capillary tubing is used to transfer drops of the pigment extract to a piece of chromatography paper.

When enough drops have been placed on the chromatography paper to make a dark green circle on the paper, it is ready to be placed into a large 25x200 test tube.

Leaf Pigment Chromatography Diagram

The large test tube has about 1/2 inch of petroleum ether in the bottom of the tube.  The pigments will immediately begin to separate.

Here is the finished chromatogram:

Pigment Chromatography FAQs:

What pigments can be seen in leaf chromatography?
Plants contain multiple pigments such as chlorophyll, carotenoids, and sometimes anthocyanins. These pigments separate based on their chemical properties when the solvent moves up the paper. 

Why do pigments appear in different bands?
Pigments travel different distances during chromatography due to differences in solubility and how strongly they bind to the chromatography paper. 

Can this experiment predict fall leaf colors?
Leaf chromatography can reveal hidden pigments, which often correspond to colors visible during autumn. 

Here is the lab that I use with my students:

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