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KMID : 0613820190290050509
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Journal of Life Science
2019 Volume.29 No. 5 p.509 ~ p.513
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Chlorophyll a/b Ratio as a Criterion for the Reliability of Absorbance Values Measured for the Determination of Chlorophyll Concentration
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Wu Guang-Xi
Lee Choon-Hwan
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Abstract
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The Beer-Lambert law states that absorbance is proportional to the concentration of a solute in a solution at a given wavelength. This linearity works for an ideal or a ‘sufficiently diluted’ solution, so this linearity is often used as a criterion for the fidelity of the absorbance value measured. In this study, we used a chlorophyll (Chl) solution, isolated from rice leaves with 80% acetone to test the use of the Chl a/b ratio as an additional criterion for checking the fidelity of measured values using four different absorption spectrophotometers: Cary4E, UV-1650PC, Versamax (a microplate reader), and NanoDrop 1,000(which can handle a 4 μl aliquot). We used Chl solutions of varying concentrations from 0.2 μg/ml to 200 μg/ml to measure absorbance values at 645 nm and 663 nm and checked the linearity first. The results indicated that the range of Chl concentrations that we can rely on based on the linearity was similar to the range in which the calculated Chl concentrations based on the measured absorbance values agreed with the known concentrations. However, some border cases or cases with very low Chl concentrations inside the fidelity range of Chl concentrations did not agree with the criterion that the Chl a/b ratio should not change after dilution of the Chl in the solution. These results suggest that the Chl a/b ratio is a better criterion for the reliability of the absorbance values measured for the determination of chlorophyll concentration than the criterion based on the linearity suggested by the Beer-Lambert law.
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KEYWORD
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Absorbance, chlorophyll, chlorophyll a/b ratio, Lambert-beer law
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