The molecule chlorophyll a has a specific shape. This shape causes wavelengths of light that we see as a dark bluish green to be reflected back. Change the shape of that molecule by adding only two atoms, making it chlorophyll b, and the light that is reflected back is now less blue and more yellow.
What does this have to do with photosynthesis? Organisms that perform photosynthesis do so by absorbing light and converting it into usable energy. This absorbed light is not reflected back. This means that the color of the pigment(s) that the organism has will determine the wavelengths of light that the organism can use. For example, plants have two types of chlorophyll molecules, chlorophyll a & b. Each of these reflects green light, meaning that green light cannot be used for photosynthesis. To help capture a bit more of the spectrum, plants have accessory pigments called carotenoids that reflect yellow, orange, and red light, absorbing a portion of the green part of the spectrum.
Chlorophylls tend to mask most other pigments in plants, so to see these other pigments, we need to separate them. You will use a process called thin layer chromatography to extract pigments from leaves, then dissolve them in a solvent.
Thin Layer Chromatography (TLC)
Below is a list of suggested materials. Spinach is suggested for the leaves, as it is easy to acquire and rich in pigment. However, consider comparing the pigments in different types of leaves for a more interesting experiment.
- Mortar and pestle
- Sand or similar material
- Soft leaves (e.g. spinach)
- Extraction solvent: 3 parts propanone to 2 parts ethoxyethane (diethyl ether)
- Capillary tubes
- Test tubes and corks
- TLC paper strips (cut to size of test tube--about 1 cm less in length)
- Chromatography solvent: 5 parts cyclohexane, 3 parts propanone, and 2 parts ethoxyethane
Procedure for Thin Layer Chromatography
- Extracting the pigments.
- Prepare your TLC strip by drawing a line across the paper in pencil 2 cm from the bottom of the strip and set aside. Important note: Handle the strip as little as possible so oils from your hands do not interfere with the process.
- Under a hood or in a well-ventilated room, put some of the leaves into the mortar with a little bit of sand (to help break the tissue apart) and some extraction solvent.
- Grind the leaves with the pestle until they have turned to mush. You may need to add more extraction solvent as it soaks into the leaf tissue.
- Thin layer chromatography.
- Pull the mush to one side of the mortar. Place the end of a fine capillary tube into the liquid, then transfer the liquid in the tube to a point in the center of your line.
- Repeat this process, drawing more liquid from the mortar, until you have a small, concentrated dot of pigment. For best results, keep the dot as concentrated in one place as possible.
- Under a hood, add 1 cm of chromatography solvent to your test tube and place this in a test tube rack (label your tube if multiple people are using the same rack).
- Add your strip to the tube with the line you drew in pencil sitting about 1 cm above the level of the solvent, then cork the tube.
- Calculating Rf values and determining polarity.
- Check on your TLC strip regularly and have a pencil with you. When the solvent has traveled up the TLC strip about 1 cm from the top of the strip, remove the strip from the test tube and draw a line in pencil at the edge of the solvent front.
- Allow the strip to dry, then measure the distance from the original line you drew (where the pigment started) to the solvent front.
- Next, measure the distance from where the pigment started to the farthest point that each pigment traveled.
- Calculate the Rf value of each pigment: divide the distance the pigment traveled by the distance the solvent traveled.
Both the chromatography solvent and the extraction solent you used are nonpolar compounds, meaning they lack residual charges. Nonpolar compounds dissolve well in nonpolar solutions, while polar compounds do not. Pigments that are more nonpolar will dissolve better in this solvent, traveling farther up the strip. More polar pigments that have residual charges (like water) will not interact much with the solvent, staying closer to the bottom of the strip. High Rf values from TLC using a nonpolar solvent means the pigment is more nonpolar. Lower Rf values mean the pigment is more polar.
Draw or tape your TLC strip and label as many pigments as you can (see the next page for more information on pigments). Record the Rf values of each pigment next to its label.
Which pigment is more polar, chlorophyll a or chlorophyll b? How can you tell?
How many pigments were present in your leaf sample?
Which pigments were the most nonpolar (least polar, highest Rf values)?
If there were polar pigments in the leaves and you used a nonpolar solvent to extract the pigments from the leaf, would they dissolve and be present in the solution you used to run your TLC experiment? How might this impact your results?