Purpose: To identify plant pigments by separation and isolation of the pigments using thin layer *paper 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. Check out this video of its use on CSI - Paper Chromatography on CSI
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
The different pigments in plants take a long time to separate, so you will need to set this up first and let it run while you are doing to other two experiments.
1. Cut a strip of coffee
filter. 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 isopropanol (acetone, fingernail polish remover will
work) in a beaker.
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 isopropanol,
but the green spot is not in the liquid. If the isopropanol touches the spot
directly, the pigment will just dissolve away.
5. Set the cup aside. The isopropanol will move up the filter paper slowly and
deposit the pigment components along the way.
Analysis
1. Assign a band number
for each pigment band - you should see greens, yellows, oranges..etc.
2. Copy the table onto your open paper and fill in the values
| 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 |
3. This experiment can be
repeated using other pigments - try lipstick, felt tip pens..etc