Mrs. Pensinger's Biology Blog: Separating Pigments Using Paper Chromatography

Our latest unit of study in biology class has been that amazing process on which we all depend- photosynthesis! Of course, we covered the basics: that photosynthesis is a chemical reaction done by certain organisms allowing them to use the energy of sunlight to convert carbon dioxide and water into glucose and oxygen. We reviewed the chemical equation for photosynthesis and emphasized the reactants needed for the reaction (carbon dioxide, water, and sunlight) and the products produced by the reaction (glucose and oxygen). We discussesd the structure of chloroplasts, the organelles in cells where photosynthesis takes place. We also began to delve into the two stages of photosynthesis, the light dependent reactions and the light independent reactions (also known as the Calvin Cycle).

We begin the unit by talking about the importance of energy for living things- we can't live without it! Where does our energy come from? Our food! Many students already know that some organisms are able to make their own food while other organisms (like us) have to consume other organisms or products of other organisms for food. We learned that autotrophs (aka producers) are organisms that can make their own food using processes like photosynthesis and chemosynthesis, and that heterotrophs (aka consumers) are organisms that must consume food. Learning these terms was a nice way to recall our science prefixes and suffixes from the beginning of the year- we remembered that auto means "self," hetero means "different," and troph means "eat or consume."

One lab that we performed during this unit focused on the pigment that is at the center of photosynthesis, chlorophyll. Chlorophyll is a green pigment found in the thylakoids, which are little disk-shaped structures inside chloroplasts. While chlorophyll is responsible for the green color in leaves of plants, its more important role is to absorb the energy from sunlight that drives the reactions of photosynthesis. The lab introduced students to a scientific technique called chromatography. There are several different forms of chromatography, but we completed a simple kind called paper chromatography. In our lab, paper chromatography allowed various pigments in different mixtures to be separated as the pigment mixtures moved along the paper. Those that were more soluble in our solvent, acetone, moved further up the paper than those pigments that were less soluble in acetone.

Students began by testing out a Mr. Sketch marker. Some groups tested black markers and some groups tested brown markers, since we hypothesized that these colors would have the most different pigments. Students placed a dot at the bottom of a piece of chromatography paper and then placed the strip of paper in a test tube with acetone. As the acetone moved up the paper due to capillary action, it carried the various pigments with it and separated them out on the paper strip. Students then set up four more strips of chromatography paper with unknown food coloring mixtures. Their goal was to see if they could determine which colors were mixed based on the pigments that separated on their chromatography strip. Many got good results and were successfully able to identify the unknown colors in each mixture.

A student adds a food coloring mixture to a strip of chromatography paper.

Examples of separation of pigments from markers and food coloring mixtures

In the second part of the lab, students were tasked with extracting the chlorophyll pigment from spinach leaves. Students were given a few leaves of spinach, along with some rocky sand and some acetone, and began to grind the leaves using a mortar and pestle to attempt to extract the chlorophyll. Once they had a small amount of liquid extract, they poured it into a test tube and placed a strip of chromatography paper down into the extract. While some worked better than others, most students were able to see various shades of green chlorophyll pigment on their chromatography strip as the liquid extract moved up the paper. Some even saw some yellow pigment toward the top!