AP Lab 5 part one: CHROMATOGRAPHY LAB
Introduction: Chlorophyllis 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) Ethyl alcohol, rubbing alcohol or acetone
Fresh spinach Scissors and Ruler
Colored leaves of plantsGlass stirring rod
Mortar and pestle100 ml beaker
Sand (optional)Pencil
10-ml Graduated cylinder 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 andimmediately mark the location of the solvent front before it evaporates as well as the different pigment colors on the filter paper. You maybe able to observe 4 or 5 pigment bands.
6) Calculate the Rf values for each pigment. See Figure 2 for instructions.
7) Repeat procedures #1-5 with a colored leaf and record data.
Data:Part One: Green Leaf:Draw a picture of your finished chromatography paper. Make sure to add color and include band numbers.
Data: Chart A: Green Leaf
Band Number / Pigment Name / Band Color / Rf Value1
2
3
4
Distance Solvent (Alcohol) Front Moved ______(mm)
Data: Part Two: Color Leaf: Draw a picture of your finished chromatography paper. Make sure to add color and include band numbers.
Data: Chart B: Color Leaf
Band Number / Pigment Name / Band Color / Rf Value1
2
3
4
Distance Solvent (Alcohol) Front Moved ______(mm)
Data Analysis: 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.