Investigation: Separation of Plant Pigments Using Chromatography
Paper chromatography is a useful technique in the separation and identification of different plant pigments. In this technique, the mixture containing the pigments to be separated is first applied as a spot or a line to the paper about 1.5 cm from the bottom edge of the paper. The paper is then placed in a container with the tip of the paper touching the solvent. Solvent is absorbed by the paper and moves up the paper by capillary action.
As the solvent crosses the area containing plant pigment extract, the pigments dissolve in and move with the solvent. The solvent carries the dissolved pigments as it moves up the paper. The pigments are carried along at different rates because they are not equally soluble. Therefore, the less soluble pigments will move slower up the paper than the more soluble pigments. This is known as developing a chromatogram.
Paper chromatography is useful for identifying unknown compounds - often used in crime scene investigations to match ink, lipstick, or colored fibers. There are many examples of chromotography at youtube.com. This set-up shows two different pen inks.
Purpose: To identify plant pigments by separation and isolation of the pigments using thin layer paper chromatography.
The distance traveled by a particular compound can be used to identify the compound. The ratio of the distance traveled by a compound to that of the solvent front is known as the Rf value; unknown compounds may be identified by comparing their Rf's to the Rf's of known standards.
Rf equation:
Rf = distance traveled by compound
distance traveled by solvent
1. Cut a strip of coffee
filter (or filter paper). Draw a horizontal line with a pencil (not pen) about half an inch from
the bottom. Place a spinach leaf on the line and roll a penny over it so that
you get a line of green pigment on the filter. Using a different part of the
leaf, roll the penny again over the same line. Repeat this process until the
line is fairly dark.
2. Put about an inch of acetone in the beaker (isopropyl alcohol will also work.)
3. Tape the top of the coffee filter strip to a pencil and balance the pencil
across the top of the beaker. See the image below for the set-up.
4. It is very important that the bottom of the filter strip is in the acetone,
but the green spot is not in the liquid. If the acetone touches the spot
directly, the pigment will just dissolve away.
5. Observe what happens to the liquid in the beaker and the spot on the filter paper. Results will take about 20 minutes.
6.Optional: Repeated using other pigments - try lipstick, felt tip pens, skittles candy..etc
Analysis
1. Assign a band number
for each pigment band - you should see greens, yellows, oranges..etc.
| Band Color | Plant Pigment | Distance (mm) | Rf (use formula) |
| Yellow to Yellow-orange | Carotene | ||
| Yellow | Xanthophyll | ||
| Bright Green to Blue Green | Chlorophyll a | ||
| Yellow Green to Olive Green | Chlorophyll b |
2. Explain how a crime lab could use paper chromatography to determine if lipstick found at a crime scene matched the lipstick of a suspect.
Other Resources on Plants
Photosynthesis with Leaf Disks – this lab uses leaf disks that float to indicate photosynthesis. Students investigate factors that affect photosynthesis. (AP Lab)
Photosynthesis and Plant Growth (virtual) – use a virtual app to show how plant growth changes in response to light color and light intensity
Leaf Transpiration – use leaves from trees to analyze how much water is lost when they are exposed to light