1. Absorption Spectrum for Plant Pigments

2. For this experiment you will be using a spectrophotometer to determine the combined absorption spectrum for the four plant pigments.
This will look much like the action spectrum for photosynthesis which is determined by plotting a graph of the oxygen release by a plant under different wavelengths of light.
The pigments are carotene, xanthophyll, chlorophyll a and chlorophyll b in a spinach leaf.
You will first need to extract a small amount of the pigments using a solvent and then analyze the extract with the spectrophotometer.

3. Procedure
To extract the pigments, tear up a few spinach leaves in to small pieces and place them in a test tube.
Add 5 mls of acetone and 5 mls of naphtha to the test tube.
Carefully grind up the leaves and solvent with a stirring rod until the solvent is a dark green.
Pour the solvent into another test tube and let it stand, layers will appear in the solvent.
The bottom layer is mostly water from the leaves, the second layer is cellular debris and the top should be a clear dark green layer of the pigments.

4. Using the Spectrophotometer
Prepare two small test tubes, one containing just the solvent and one containing the solvent and pigments.
To two small test tubes add 2.5 mls of acetone and 2.5 mls of naphtha. From the test tube with the extract carefully remove just the dark green top layer using a pipet. Add 10 drops of this solution to one of the small test tubes.
This is the tube that will be used to produce the action spectrum the other is used to calibrate the machine during testing.

5. Using the Spectrophotometer
Prepare two small test tubes, one containing just the solvent and one containing the solvent and pigments.
To two small test tubes add 2.5 ml of acetone and 2.5 ml of naphtha.
From the test tube with the extract, carefully remove just the dark green top layer using a pipet.
Add 10 drops of this solution to one of the small test tubes. This is the tube that will be used to produce the action spectrum the other is used to calibrate the machine during testing.

6. The spectrophotometer will tell you the amount of visible light absorbed by the pigment. For each reading the machine must be calibrated using just the pure solvent. Why must you constantly recalibrate the machine when you change wavelengths???

7. Calibrating the Spectrophotometer
The two knobs on the front are for calibration:
The left knob is to zero the meter reading with no sample in the machine and top door is closed.
The right knob is to set the meter to full scale with a test tube of solvent is in the machine and the top door closed.
Once the machine is calibrated you can place your sample in the machine and take a reading… but every time you change wavelengths you must recalibrate.

8. Take Absorption Readings
Measure 380 nm to 600 nm with the filter lever turned to the left
Measure with the filter lever turned to the right

9. Plot data on graph; label colors (words/colors)
Answer questions

10. Plant Pigment Separation
Cut several strips of filter paper using paper cutter
Put some solvent or a combination of solvents in the bottom of the plastic tube
Draw a line with a pencil on the origin by the notch

11. Plant Pigment Separation
Put your filter paper + pigments in the tube and monitor carefully
Remove the strip when the solvent front reaches 1 cm from top
Identify plant pigments, and calculate Rf values.
Try at least 3 different solvent systems

12. Write a single short lab report (as a group), including your chromatography paper taped to the report.