Chlorophyll Content
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Materials
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Chloroplast suspension from Exercise 8.1
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Acetone
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Spectrophotometer and tubes
- Clinical centrifuge and tubes
Procedure
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Pipet 1.0 ml of a chloroplast suspension into a 15 ml centrifuge tube and add 8.0 ml of acetone. Mix thoroughly.
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Add 1.0 ml of distilled water, mix again and centrifuge at 1000 xg for 3-5 minutes.
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Turn on a spectrophotometer, adjust the wavelength to 652 nm and blank the instrument with water.
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Measure the absorbance of the supernatant from step 2.
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Calculate the amount of total chlorophyll in 1.0 ml of the chloroplast suspension. 2 The amount of chlorophyll is givenby the equation:
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Calculate the dilution factor for your original chloroplast suspension so that when diluted with suspension solution, the final concentration of chlorophyll will be 0.01 mg/ml.
All subsequent work with chloroplasts will use the diluted suspension so that the criterion of step 6 is met.
- Use a hemacytometer to determine the number of chloroplasts per ml of any diluted suspensions.
<center> <table> <tbody> <tr> <td> <font>Absorbance of acetone extracted chlorophyll solution = ___________<br /> Calculated concentration of chlorophyll = ____________mg chlor./ml<br /> Dilution factor for suspension to yield 0.01 mg chlorophyll/ml = ___________<br /> Chloroplasts/ml of suspension so diluted = ___________<br /> </font></td> </tr> </tbody> </table> </center> <center> <p> </p> </center>
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