Leaf Pigment Chromatography Lab with Spinach for Photosynthesis

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.

Connecting Leaf Pigment Chromatography to Photosynthesis

Leaf pigment chromatography works especially well during a photosynthesis unit because students can see that leaves contain more than one photosynthetic pigment. Separating chlorophylls and carotenoids gives students a visual way to explore how different pigments absorb light and contribute to the process of photosynthesis. I like using the lab to move the discussion beyond simply thinking of leaves as “green” and toward understanding the different pigments involved in capturing light energy.

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:

Click image to view in my TpT store.

Paper Chromatography Explained: Diagram, Lab Ideas, and Classroom Tips

Looking for a low-prep, high-impact paper chromatography experiment to spark curiosity in your middle or high school science classroom? Paper chromatography is a classic lab technique that allows students to separate and analyze mixtures through hands-on investigation. In this paper chromatography experiment, students observe how different substances move along chromatography paper at different rates, and a simple diagram helps illustrate how solubility, polarity, and molecular properties affect separation. Whether you are introducing the scientific method, studying photosynthesis, or exploring real-world applications of chemistry and biology, paper chromatography is a versatile and visually rewarding lab to include in your curriculum.

What Is Paper Chromatography?

Paper chromatography is a simple chromatography technique used to separate the components of a mixture. This method works by taking advantage of differences in solubility and molecular interactions, allowing substances to be identified based on how far they travel up a strip of paper in the presence of a solvent. In the real world, chromatography is an essential analytical tool used in fields such as forensics, toxicology, and pharmacology. Police labs use chromatography to detect drugs and toxins in blood and urine samples, making this paper chromatography lab a meaningful way to connect classroom science to real-world STEM careers.

If you are looking for a ready-to-use paper chromatography lab for your classroom, you can find the complete student instructions, data tables, and teacher notes in my Paper Chromatography Lab resource on Teachers Pay Teachers.

Paper Chromatography Diagrams

These paper chromatography diagrams show how a solvent moves up the chromatography paper, carrying dissolved substances with it. Different components of the mixture travel at different rates based on their solubility and attraction to the paper, resulting in visible separation. This visual helps students better understand how paper chromatography works during the experiment. 

Paper Chromatography Experiment Overview

👉 The process is straightforward but packed with learning potential. A small dot or line of a sample (like ink or plant pigment) is placed near the bottom of a strip of chromatography paper. This strip is then placed vertically into a container with a shallow layer of solvent. As the solvent travels up the paper by capillary action, it carries the components of the mixture with it.

Here’s why the mixture separates:

• Solubility: More soluble substances dissolve better in the solvent and move farther up the paper.
• Molecular Weight: Smaller molecules tend to move more quickly than larger ones.
• Polarity: The cellulose in the paper is polar. More polar substances stick to the paper and don’t travel as far, while non-polar substances move more freely.

Chromatography Lab Ideas for the Biology or Physical Science Classroom

💧 Ink Separation

A fun and accessible introduction to chromatography is separating black ink from markers. Many black inks are actually mixtures of several colors. When placed in water, water-soluble inks spread apart into bands of dye. Not all markers will work the same way. Permanent inks may require rubbing alcohol or another solvent to separate. This gives students the opportunity to test variables and compare results across different solvents and ink types.

🍃 Plant Pigment Separation

This is a favorite in high school biology, especially during units on photosynthesis or plant biology. Students can extract pigments from leaves and discover that the color they see is actually a mix of chlorophyll-a, chlorophyll-b, xanthophyll, and carotene.

Fall is a perfect time to collect a variety of leaves from trees around your school. But if you’re teaching in winter, spinach leaves from the grocery store work just as well.

Materials for a Paper Chromatography Experiment

You don’t need a fancy setup to get started. Here's a basic chromatography kit:

• Chromatography paper (filter paper or coffee filters can work in a pinch)
• Small containers (test tubes, jars, or beakers)
• Solvents (water, rubbing alcohol, acetone, or other safe household solvents)
• Samples to test (leaves, markers, food dyes, etc.)
• Pencils, rulers, and scissors

Tip for teachers: Always test your materials and solvents in advance. Be sure to follow proper lab safety protocols, especially if using alcohols or other flammable substances.

Paper Chromatography Lab for High School Biology and Chemistry

Paper chromatography is one of the most effective chromatography labs for high school biology and chemistry classes because it allows students to visualize molecular differences directly. This chromatography experiment helps students understand key concepts such as polarity, solubility, intermolecular forces, and molecular structure.

In high school biology, paper chromatography is commonly used to separate plant pigments during photosynthesis labs. In chemistry classes, students use chromatography to analyze ink, food dyes, and other mixtures. Because the setup is simple and inexpensive, this chromatography lab is ideal for introducing experimental design, controlled variables, and data analysis.

This paper chromatography lab also helps students connect abstract molecular concepts to real, visible results, making it one of the most memorable chromatography activities in the science classroom.

What Solvent Should You Use?

The answer depends on the solubility of your sample. Water works well for many inks and food dyes. For permanent markers or plant pigments, isopropyl alcohol or acetone may be more effective.

Pro Tip: Encourage students to design their own investigations. They can test multiple solvents on the same type of ink or compare different brands of markers to determine which separates most clearly.

Why Use Paper Chromatography in the Biology Lab?

Paper chromatography gives students hands-on experience with:

• Designing and conducting controlled experiments
• Observing molecular behavior and physical properties
• Making qualitative and quantitative observations
• Connecting lab techniques to real-world applications

It’s also a visually exciting lab. Watching vivid colors separate into bands on a strip of paper never gets old, and makes for excellent lab notebook sketches!

Check out this post on my blog, Leaf Pigment Chromatography, for additional photos and information. 

You may also be interested in my osmosis lab using dialysis tubing and my potato osmosis experiment, which help students understand diffusion, osmosis, and molecular transport in cells. Check out these blog posts:

• Potato Osmosis Experiment
• Osmosis Lab Using Dialysis Tubing

Frequently Asked Questions About Paper Chromatography

What is chromatography used for?
Chromatography is used to separate and analyze mixtures by observing how different substances move through a medium. Scientists use chromatography in fields such as forensics, medicine, environmental science, and food testing.

Why does paper chromatography work?
Paper chromatography works because different substances have different attractions to the paper and the solvent. These differences cause components of a mixture to travel at different speeds, resulting in separation.

What can students learn from a paper chromatography experiment?
Students learn key lab skills such as experimental design, data collection, observation, and analysis while exploring concepts like solubility, polarity, and molecular interactions.

How do you perform a paper chromatography lab in the classroom?

To perform a paper chromatography lab, students place a small sample of ink or plant pigment near the bottom of chromatography paper and suspend the paper in a solvent. As the solvent travels upward, it separates the mixture into its components based on solubility and polarity. Students can measure how far each component travels and calculate Rf values to analyze the results and compare substances.

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Ready to Try This With Your Students?

These chromatography labs have been classroom tested and include complete student instructions, answer keys, and teacher guidance for easy implementation. If you're looking for classroom-ready chromatography labs, I have two student-tested resources that include everything you need: detailed procedures, printable lab handouts, answer keys, and grading rubrics.

🔬 Matter Lab Stations: Ink Separation – Perfect for introducing lab skills and the scientific method in both middle and high school.

🌿 Plant Pigment Chromatography Lab – Ideal for exploring photosynthesis and leaf pigments in your high school biology class.

Both labs are designed to save you prep time while providing meaningful, standards-based instruction. You can find them in my Teachers Pay Teachers store.