CHROMATOGRAPHY LAB
Introduction: Chlorophyll is a chemical pigment that asborbs sunlight for
plants. It is located in the chloroplasts of eukaryotic plant cells. Chlorophll
often hides the other pigments present in leaves. In autumn the
chlorophyll breaks down, allowing xanthophyll (yellow) and carotene
(orange), chlorophyll a (blue-green), chlorophyll b (yellow-green), and
newly made anthocyanin (reds, blues and purples), to show their colors.
Paper chromatography is a useful technique for separating and identifying
pigment and other molecules from cell extracts that contain a complex
mixture of molecules. The solvent moves up the paper by capillary action,
which occurs as a result of the attraction of solvent molecules to the paper
and the attraction of the solvent molecules to one another. As the solvent
moves up the paper, it carries along any substances dissolved in it. The
pigments are carried along at different rates because they are not equally
soluble in the solvent and because they are attracted, to different degrees,
to the fibers of the paper through the formation of intermolecular bonds,
such as hydrogen bonds.
Materials:
Safety goggles
Chromatography paper (or filter paper)
Fresh spinach
Colored leaves of plants
Mortar and pestle
Sand (optional)
10-ml Graduated cylinder
Ethyl alcohol, rubbing alcohol or acetone
Scissors and Ruler
Glass stirring rod
100 ml beaker
Pencil
Tape
Procedure:
1. Obtain a 250 mL beaker which has about 2 cm of solvent (alcohol)
at the bottom.
Cover the beaker with aluminum foil to prevent the vapors from
spreading.
2. Cut a piece of filter or chromatography paper which will be long
enough to
reach the solvent. Draw a line about 3.0 cm from the bottom of the
paper.
3. Use a quarter to extract the pigments from spinach leaf cells. Place
a small
section of leaf on the top of the pencil line. Use the ribbed edge of the
coin to crush
the leaf cells. Be sure the pigment line is on top of the pencil line.
Use a back
and forth movement exerting firm pressure through out.
4. Place the chromatography paper in the cylinder. Do not allow the
pigment to
touch the solvent (alcohol).
5. Cover the beaker. When the solvent is about 1 cm from the top of
the paper,
remove the paper and immediately mark the location of the solvent
front before it
evaporates as well as the different pigment colors on the filter paper. You may be able to observe 4 or 5 pigment
bands.
6. Repeat procedures #1-5 with a colored leaf and record data.
Data: Part One: Green Leaf: Draw a picture of your finished chromatography paper in your lab report. Make sure
to add color!
Data: Chart One: Green Leaf
Band Number Pigment Name
1
2
3
4
Band Color
Data: Part Two: Color Leaf: Draw a picture of your finished chromatography paper in your lab report. Make sure
to add color!
Data: Chart Two: Color Leaf
Band Number Pigment Name
1
2
3
4
Band Color
Distance Solvent (Alcohol) Front Moved _________________ (mm)
Conclusion: Answer using complete sentences.
1. How could you differentiate (tell the difference) between chlorophyll a and chlorophyll b?
2. Which pigments are present in the smallest amounts in the leaf?
3. Which pigments are present in the greatest amount?
4. What factors are involved in the separation of the pigments?
5. What adaptive purpose do different colored pigments serve for a plant (why does this help the plant survive)?
Explain.
6. Why do some pigments move farther up the paper than others? Explain.
7. Your friend performed this chromotography technique with a piece of onion. As he ran his test, he noticed there
was no chlorophyll on his filter paper. Why is this so?
8. What data was used to make the conclusion that chlorophyll was present in your leaves? Explain.
Extra Credit/Honors Challenge: Repeat the procedure using the black ink in your pen. Record a picture of your
test paper and identify the pigments in a black pen. Record the results in your lab report. Clearly title it Extra
Credit!