Properties of Chlorophyll in Plants
Properties of Chlorophyll in Plant
1. Measuring the Concentration of Chlorophyll in a Leaf
For this work B &L Spectronic 20 spectrophotometer should be used to measure the optical density of a chlorophyll solution in order to determine the concentration of chlorophyll in a leaf.
Before proceeding, become familiar with the use of the B &L Spectronic 20.
Measure 1 g of fresh leaf tissue and cut the leaves into small pieces (about 1 mm wide) with scissors or razor blade. Extract the pigment by grinding the cut tissue for 5 minutes in 100 ml of 85% acetone in a mortar and pestle.
Transfer the homogenate to a Buchner funnel fitted with Whatman No. 1 filter paper and filter the extract using the vacuum units as demonstrated by the instructor. Transfer the filtered extract to a 100-ml volumetric flask and make up to volume with 85% acetone.
Measure the optical density (absorbance) of the extract with the B &L Spectronic 20. Measure optical density at both 663 nm and 644 nm. These are positions in the spectrum where maximum absorption by chlorophyll a and b occur. The concentration of chlorophyll a and b, in mg per g of tissue, is calculated by the formula:
mg chlorophyll a/g tissue = 1.07 (O.D. 663) - 0.094 (O.D. 644)
mg chlorophyll b/g tissue = 1.77 (O.D. 644) - 0.280 (O.D. 663)
The constants used in these calculations have been determined empirically (Arnon, D. I., "Copper enzymes in isolated chloroplasts; polyphenol-oxidase in Beta vulgaris," Plant Physiol. 24:1-15, 1949; Koski, V., "Chlorophyll formation in seedlings of Zea mays L.," Arch. Biochem. Biophys. 29:339-343, 1950).
Save the chlorophyll extract for use in parts 2 and 3.
2. Measuring the Absorption Spectrum of Chlorophyll a and b
Dilute some of the chlorophyll extract from Part 1 with 85% acetone so that the diluted solution transmits 30 to 40% of the light at 640 nm. If the transmittance is already greater than 40% then skip the dilution step. (Remember to set the instrument at 100% transmittance with-a tube containing only 85% acetone before readings are made with the solution of pigments.) Use matched cuvettes, one for the diluted pigment system and one for the blank of 85% acetone.
Using the diluted solution of chlorophyll a and b, measure the transmittance every 10nm from 400nm to 700nm (ie. 400, 410, 420, etc.). Be sure to reset the instrument to 100% transmittance, with the cuvette containing only 85% acetone, before making each reading.
Convert all transmittance values to otical density (absorbance).
Plot wavelength on the abscissa and optical density on the ordinate, and construct a graph of the absorption spectrum of chlorophyll a and b.
Absorbance of light of chlorophyll a(green) and b(red)
3. Optical Density as a Function of Concentration of the Pigment
Quantitatively dilute 50 ml of the chlorophyll extract from Part 1 with 85% acetone so that the diluted solution has a transmittancy of 10% at the wavelength of maximum absorption (as determined in Part 2). Do this by adding measured amounts of 85% acetone to the sample of the original extract. After each addition, remove a sample and measure its transmittance. Continue until the transmittance reaches 10%. If the transmittance is already 10% or higher do not dilute it further. Keep track of how much acetone you add to the flask. Using the values for chlorophyll concentration determined in Part 1, calculate the concentration of pigment in this diluted solution.
Using the diluted solution having about 10% transmittancy, prepare five cuvettes as follows using 85% acetone:
After setting the instrument to 100% transmittancy with a solvent blank prior to each reading, measure the optical density of the solution in each tube at the wavelength of maximum absorption for the pigment.
Construct a graph of optical density as a function of concentration of the pigment.
According to the Beer-Lambert Law, optical density = ebC, where e is a constant called the extinction coefficient, b is the length of the light path through the pigment solution, and C is the concentration of the pigment. (Because b was the same during all of your measurements, it can be ignored in computations.) Comparing your observations with the equation for optical density, look at the shape of the curve in your graph if it fits the equation