I am planning to compute for the chlorophyll content of a certain plant using the UV-Vis Spectrophotometer but I don't know how to formulate an equation regarding it. I've read different researches containing equations like this but I've observed that as the wavelengths vary, the numerical coefficients beside the variable of absorbance of a certain wavelength also vary. This is why I think there's a general equation for this.
Also, where could I get a standardized method for chlorophyll determination of plants using the UV-Vis Spectrophotometer?
if you have only one component leading to light extinction, measurement at one wavelength is sufficient. When you expect interference due to a second component or simply by turbidity, measurement at a second wavelength or recording differential spectra can be applied.
For the 2 component you can write down 2 equations for measured extinction with 2 unknowns and solve it for concentration of chlorophyll.
This BBA ref is authoritative but care must be applied to chl pheophytin degradation upon extraction which must be prevented by Na ascorbate and Pheo check, http://www.chem.ucla.edu/dept/Faculty/merchant/pdf/Porra_1989.pdf
Here is the reference I use. It covers spectrophotometers with different resolutions and different solvents for extracting chlorophyll. The extinction coefficients are supplied for these various options.
Dear Bryan,
You don't mention if you want to measure chlorophyll contents of leaves or pigment extractions, i.e. chlorophylls dissolved in various solvents.
In case of solutions, you must consider that the different solvent molecules modify the delocalized electron system of chlorophyll in different ways, i.e. they cause differences in the positions of the absorption maxima and in the extinction coefficients. In fact, an absorption spectrum is the wavelength dependence of the extinction coefficient values. You can use one wavelength measurement only if you are 100% sure that your sample has only one pigment and if you know the absorption maximum of that pigment. Titus explained correctly that you may have cjlorophyll-a and chlorophyll-b or breakdown products. An additional problem is the baseline distortion: if you measure only one single absorbance value, you never know what part of this value is the real pigment absorption and what part is caused by light scattering - baseline distortion.
The measurement of intact leaves is popular in ecology but it cannot give analytically correct results.
Best greetings,
Bela Böddi
Using a spectrophotometer to measure chlorophylls in solvents is not difficult and you can look at my papers on it or look at the papers by Porra. I gather that you want to measure Chl in vivo. That is much more difficult. For unicellular algae you can work out a correlation of A680 in 60% sucrose vs extractable Chl a. But every alga is different. You can get a very close linear relationship. For macrophytes and vascular plants it is much more difficult. You need an integrating sphere called a Taylor sphere. Unfortunately a Taylor sphere often costs as much as the spectrophotometer itself and in some countries they are very hard to find..
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