Chromatography and Fall

I haven't done any work in chromatography since high school when I was working with benzene aniline dyes. Nevertheless, here is a cool article and hands-on procedure for using chromatography and Fall leaves from Scientific American.

"Find the Hidden Colors of Autumn Leaves"

A chemistry challenge from Science Buddies

October 20th, 2011

Scientific American

Introduction

Have you ever wondered why leaves change from green to an amazing array of yellow, orange and red during the fall? Leaves get their brilliant colors from pigments made up of various size, color-creating molecules.

During the warm, sunny months, plants use their leaves to turn sunlight into food energy, a process called photosynthesis. This primarily uses a pigment that reflects green light, which gives the leaves their characteristic color.

In autumn, when colder, shorter days arrive, many kinds of trees no longer make food energy with their leaves and, consequently, no longer need the green pigment. The leaves' other pigments, some of which were already there during summer, become visible. Uncover these hidden colors of fall by separating plant pigments with a process called paper chromatography. What colors will you see?

Background

There are many types of pigments in plant leaves. Chlorophyll makes them green and helps carry out photosynthesis during warm, sunny months. As fall arrives and the green, food-making color fades, other pigments such as yellow, orange and red ones become more visible.

Xanthophylls are yellow pigments, and carotenoids give leaves an orange color. Photosynthesis also uses these pigments during the summer, but chlorophyll, a stronger pigment, overpowers them. These pigments take more time to break down than chlorophyll does, so you see them become visible in fall leaves. They're also found in carrots, daffodils, bananas and other plants that have these vibrant colors. There are also anthocyanins, intense red pigments that aren't made during the summer, only appearing with the final group of the fall colors. These molecules also give the red hue to apples, cranberries, strawberries and more.

Although a leaf is a mixture of these pigments, you can separate the colors using a method called paper chromatography. This process dissolves the pigments and allows them to be absorbed by a strip of paper. Larger molecules have a harder time moving in the woven paper and get trapped in the paper first, whereas smaller ones travel farther along the paper. This process separates the mixture of pigments by molecular size—and by color.

Materials

• Leaves at different stages of turning colors (the more the better—about 10 of each color is best)
• Scissors
• Strong, sturdy drinking glasses (three to four)
• Rubbing alcohol (isopropyl alcohol)
• Wooden spoon or another wooden utensil with a blunt end for crushing leaves
• Fork
• Very small bowls or tea-light candleholders (three to four)
• Strong, white, heavyweight, ultra-absorbent paper towels
• Ruler
• Pencil
• Toothpicks
• Plate (or other surface to protect working area from stains)
• Tall glass jars, such as mason jars (three to four)
• Clothespins or large paper clips (nine to twelve)

Preparation

• Collect some leaves that are at different stages of color change during the fall, preferably from the same tree.
• Separate your leaves into distinct groups arranged by color, with about 10 large leaves per group. Separating them into green, yellow and red piles may be easiest.
• Prepare paper towel strips, making three to four strips for each group of leaves. Cut up a strong, thick paper towel into long, one-inch-wide strips. They should be long enough to touch the bottom of the tall glass jars or mason jars and still extend over the top. With a pencil, gently draw a line one inch from the bottom of each strip.

Procedure

• Cut the leaves into small pieces with scissors. Put each group of leaves into the bottom of a drinking glass.
• Add one tablespoon of rubbing alcohol to each glass.
• Crush the leaves into the rubbing alcohol using the blunt end of a wooden spoon for about five minutes, until the solution is dark. How has the color of the alcohol changed?
• Let the solution sit for 30 minutes in a dark place indoors.
• Use a fork to remove any leaf pieces from the solutions and discard these, while leaving the liquid in the glass.
• Pour each solution into a very small bowl, and leave it in a dark place indoors to allow more of the alcohol to evaporate. You will be ready for the next step when you stir your solutions with a toothpick and they seem thicker.
• Thoroughly stir each colored solution with a toothpick, using a different toothpick for each solution so as not to mix the colors.
• Using a toothpick for each color, smoothly and evenly "paint" some of each solution across a paper towel strip on the pencil line you drew. Because some plant pigments can stain, you should do this on a plate so that the color will not stain your work surface. For each color, do this using a total of three to four strips.
• Allow the strips to dry.
• While the strips are drying, pour enough rubbing alcohol into each glass jar to just cover the bottom. Prepare one jar for each color solution.
• With the dry strips, carefully put the pigmented end into the jar until the strip just touches the alcohol. Drape the top of the strip over the jar's opening and secure it with a clothespin. Make sure that each strip is not touching the jar's sides, but only contacts the jar where it is secured. Place and secure strips from the same solution into the same jar, but keep them from touching each other.
• Let the glasses sit for 30 minutes and watch the paper strips. What is happening to the color of the paper strips?
• When one of the colors reaches the top of a strip, remove all strips and let them dry.
• Look at the different dried strips. How are the colors in the strips different? Do strips from different color solutions have unique colors, shared colors or both?
• Look at the order in which the colors appear on the different strips. Is the same color on the same place in different strips or is it in a different place? Do the colors appear in the same separation order or in different orders on each strip?
• Tip: If your chromatography comes out pale, try using more leaves, cutting them up into smaller pieces and/or "painting" more of your solution onto the pencil line on the paper towel.
• Extra: You can use this same procedure to compare the color molecules in many different plant sources. For example, you could try red cabbage, blueberries, carrots, beets, spinach, flowers or other intensely colored plants. How do their mixtures of color molecules compare?
• Extra: If you find a tree with a wide range of colors, you can repeat this procedure using leaves at more intermediate stages of change. An especially good source of a wide variety of leaf colors is aspen trees.

Observations and results

Were you able to see multiple bands of color on your test strips? Did you see that some of the bands present were different for the different color solutions used?

Even though a plant leaf looks like it is mostly one color, it is actually made up of a mixture of pigment molecules. In this procedure, paper chromatography separated the pigments by the size of their molecules. Consequently, you should see different colors at different locations as you go along one of the paper towel strips, and the order in which the colors appear should be roughly the same among the different color solutions you tested.

What are the different bands of color on the test strips? These are the different pigments in the leaves. The ones you may see on your paper towel strips are: green chlorophylls, yellow xanthophylls, orange carotenoids and red anthocyanins. Pigments with larger molecules generally stay near the bottom of the strip, where the solution was first "painted" onto the pencil line, because it is harder for them to travel up through the paper towel's woven fiber. Smaller pigments can more easily traverse the paper towel and, consequently, they usually travel farther up the strip.

Because the color of the leaf is dependent on the mixture of pigments within it, different colored leaves will display different colors on their paper towel strips. For example, very green leaves may not have any red colors (anthocyanins) on their strips.

Cleanup

Because some plant pigments can stain, be careful not to spill your colored solutions when throwing them away.

More to explore

"What Causes the Leaves on Trees to Change Color in the Fall?" from Scientific American​

"Why Leaves Change Color," from S.U.N.Y. College of Environmental Science and Forestry E-Center

"Autumn Foliage Color," from The Fisher Museum, Harvard Forest, Faculty of Arts and Sciences of Harvard University; and Florida International University, Department of Biological Sciences

"Paper Chromatography Resources"

"Paper Chromatography: Basic Version"