This post originally appeared on the blog Amy Brown Science.

What is paper chromatography? Paper chromatography is a simple and cost-effective method of separating the components of a mixture. By using this procedure, the individual parts of the mixture can be identified after the components of the mixture have been separated. Understanding the concept of chromatography is extremely relevant to the students in our science classes. Chromatography is used in crime scene testing to analyze blood and urine samples for poisons, toxins, or alcohol levels. It is also used to test for doping or performance enhancing drugs in athletes. While the chromatography techniques used in these instances is more advanced than can be carried out in a school lab, we can utilize the technique of paper chromatography to separate the colored chemicals or substances in a mixture.

How does it work? In the paper chromatography technique, a small dot or line of the mixture is placed onto a strip of chromatography paper or filter paper. The paper is then placed in a jar, beaker, or large test tube containing a small amount of solvent. As the solvent rises up the paper, it meets the sample mixture, and carries the sample mixture along with it. Along the way, the different components of the mixture are separated out at different levels on the paper.

Several factors explain why the different parts of the mixture separate out as they do:

Chromatography is used in crime scene testing to analyze blood and urine samples. Here's how to bring this fascinating method into your science lab.

  • Solubility: If the components of the mixture are soluble in the solvent being used, the mixture will dissolve as the solvent front moves through it, and the mixture will be carried up the chromatography paper strip as the solvent travels. Some of these substances in the mixture will likely be more or less soluble than others.  The more soluble substances will move faster and to a greater distance than those that are less soluble.
  • Molecular Weight: The substances of lighter molecular weight will move higher up the paper than those substances having a higher molecular weight.
  • Interactions between the paper and the mixture: The fibers in the paper are made of cellulose, which is a polar substance. The components within the mixture may have different degrees of polarity. Substances that are nonpolar will travel farther up the paper. Substances that are more polar will bond with the cellulose paper more quickly, and will not travel as far.
How can this be used in middle and high school experiments? A very popular lab activity to carry out in middle and high school labs is the separation of ink mixtures. The ink in a black, water-soluble marker is actually a mixture of several different colors. The ink is simply applied to the chromatograph paper and the tip of the paper is placed in a solvent. Many common inks are water soluble. This means that you can use water as the solvent in order to separate the mixture. If the ink you are testing does not separate out using water, it may be a “permanent” ink. As a result, you will need to use a different solvent such as rubbing alcohol or acetone.  
 
What materials are needed to do this?  This experiment can be done with very simple materials.  
•  Chromatography paper or filter paper
•  Some sort of container (jar, beaker or large test tube) 
•  Solvent
•  A mixture that can be separated
 
What is the procedure for setting up the experiment? Chromatography is used in crime scene testing to analyze blood and urine samples. Here's how to bring this fascinating method into your science lab.

Place a small amount of solvent in a beaker or glass jar. (Keep reading below for possible solvents you can use.) Take a colored marker and make a dot of ink approximately 1/2 inch from the bottom of the paper strip. You will need to suspend the chromatograph paper over the beaker. This can easily be done by taping the paper to a glass stirring rod or pencil as seen in the photo.

Make sure that the tip of the chromatography paper touches the solvent in the bottom of the container, but also make sure that the dot of ink is ABOVE the solvent level in the container.  

The separation of pigments in the ink mixture will begin immediately.  Allow enough time to elapse until a clear separation of the mixture can be viewed.

Chromatography is used in crime scene testing to analyze blood and urine samples. Here's how to bring this fascinating method into your science lab.

What solvent should I use? The solvent used depends upon the solubility of the mixture you are trying to separate.  I have had success with water, isopropyl alcohol, ethyl alcohol, acetone, and petroleum ether.  Before doing the lab with your students, experiment with various solvents to see which works best for you.

What other mixtures can be separated in this way? My favorite use of paper chromatography is to separate the pigments found in leaves.  A green-pigmented leaf can easily be separated to show that it contains chlorophyll-a, chlorophyll-b, xanthophyll and carotene.  Since our school is surrounded by trees and shrubs of all types, I have students run several chromatograms using different leaves and compare the results. During the winter, using fresh spinach from the grocery store will yield excellent results.
Chromatography is used in crime scene testing to analyze blood and urine samples. Here's how to bring this fascinating method into your science lab.
Chromatography is used in crime scene testing to analyze blood and urine samples. Here's how to bring this fascinating method into your science lab.
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 25 x 200 mm test tube.

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:

Chromatography is used in crime scene testing to analyze blood and urine samples. Here's how to bring this fascinating method into your science lab.
 
Paper chromatography is a very cost-effective teaching tool for science teachers. The benefits of teaching this lab technique are many: It is relevant to everyday life, requires students to use their critical thinking and problem solving skills, and allows students to practice and reinforce their lab techniques. Click these links to view both the ink mixture separation and the leaf pigment separation labs in my TpT store.
Chromatography is used in crime scene testing to analyze blood and urine samples. Here's how to bring this fascinating method into your science lab.
 
 
Chromatography is used in crime scene testing to analyze blood and urine samples. Here's how to bring this fascinating method into your science lab. Amy Brown resides in Tennessee. She is married to her husband of 35 years and has two incredible daughters. Amy loves nature, the environment, and tries to spend as much time as possible outdoors. She loves to travel with her family to national parks and other outdoor locations, trips which her family lovingly refer to as “Mom Adventures.” Amy has 31 years of teaching experience in the biology and chemistry classrooms. She joined TpT as a teacher-author in 2006, shortly after the TpT site was launched. Amy’s “hands-on” approach to teaching science instills a love of science and nature in her students and encourages them to make science a part of their everyday lives. In Amy’s words, “I just love to get kids hooked on science!”

For more teaching tips and ideas about how to make science come alive in your classroom, visit Amy on her blog, AmyBrownScience.com. You can also keep up with Amy’s activities on Pinterest, Facebook, and Instagram.