CHROMATOGRAPHY LAB
INTRODUCTION:
Chlorophyll often hides the other pigments present in leaves. In autumn the
chlorophyll breaks down, allowing xanthophyll (yellow) and carotene (orange),
chlorophyll a (yellow-green), chlorophyll b (blue-green), and newly made
anthocyanin (reds, blues and purples), to show their colors.
The mix of pigments in a leaf may be separated into bands of color by using
a technique called chromatography. Chromatography means "color
writing”. Chromatography involves the separation of mixtures into individual
color components. Using this technique, the components of a mixture in a liquid
medium are separated. The pigment is placed on chromatography paper 1 or 2
cm from the bottom. The separation occurs when the chromatography paper is
placed barely touching a solvent. The pigment is wicked up and carries the
various colors travel up the paper. The paper holds the substances by absorption;
capillarity pulls the substances up the paper at different rates. Pigments
are separated on the paper and show up as colored streaks. The pattern of
separated components on the paper is called a chromatogram.
MATERIALS:
Safety goggles
Chromatography paper (or filter paper) about 3 cm x 10 cm Ethyl
alcohol, rubbing alcohol or acetone
Fresh spinach
Scissors and Ruler
Fresh leaves of plants
Glass stirring rod
Mortar and pestle
100 ml beaker
Sand (optional)
Pencil
10-ml Graduated cylinder
Tape
PROCEDURE:
Work in pairs. Leaves should be identified before beginning.
CAUTION: Chromatography solvents are flammable and toxic. Have no open flames; maintain good ventilation; avoid inhaling fumes.
1. Cut a strip of filter paper or chromatography paper as directed. Draw a faint pencil line 1-2cm from the bottom of the strip. The sides of the strip should not touch the glass.
2. Tear assigned leaf into pieces about the size of a postage stamp. Put them into a mortar along with a pinch or two of sand (to help with grinding). Add about 5 ml ethyl alcohol to the leaf pieces. Crush leaves with the pestle, using a circular motion, until the mixture is finely ground. The liquid in the pestle is called the pigment extract.
3. Use a glass rod to touch a drop of the pigment extract to the center of the pencil line on the paper strip. Let it dry. Repeat as many as 20 times, to build up the pigment spot. Each dot must dry before another drop is added. The drying keeps the pigment dot from spreading out too much.
4. Pour 10 ml chromatography solvent into the beaker. Tape the chromatography paper to the middle of the pencil. Rest the pencil across the top of the beaker, adjusting it so that the paper just touches the solvent (but not the sides of the beaker). The pigment dot must be above the level of the solvent. Watch the solvent rise up the paper, carrying and separating the pigments as it goes. Allow the paper to stay in the solvent for 5 minutes. At the end of 5 minutes, remove the paper and let it dry. Observe the bands of pigment. Identify and label the pigment bands on the dry strip. Record the species, external color, and chromatogram pigments in the DATA TABLE of your report sheet.
Rf = Distance moved by the pigment / Distance moved by the solvent
Measure the distance in cm from the starting point (pencil line) to the center of each pigment band. Then measure the entire distance traveled by the solvent. Remember, the starting point for the solvent is also the pencil line and the ending point for the solvent is the top edge of the paper. Do the required divisions and record your Rf values in the DATA TABLE of your report sheet.
6. Wash the mortar and pestle thoroughly, using a little alcohol to remove any remaining pigment.
DATA TABLE:
