How to determine chlorophyll content from spectrophotometry data (absorbance vs wavelength)?

Question

I boiled spinach solution (spinach leaves and 90% methanol) in solutions of varying $\mathrm{p}H$. I used a spectrophotometer to obtain wavelength ($\lambda$) vs absorption, and want to investigate chlorophyll degradation between the different $\mathrm{p}H$ and determine which $\mathrm{p}H$ would be most ideal to minimize chlorophyll degradation. I want to check if I am analyzing my data right: Can I simply take the $\lambda_{max}$ for each measurement and use that in the Beer Lambert law to determine the concentration? I know the light pathway (cuvette width) and absorbance (from spectrophotometer), but do not know the molar absorptivity. I was thinking that since its value would be a constant, I would leave it as with whatever concentration I determine from the calculation, and it would still be comparable. However, if I use this methodology for analysis, I would be ignoring the fact that the wavelengths at which the $\lambda_{max}$ is observed is different for each sample. How would I incorporate that because that is surely important to mention and analyze as well.

Or could I just analyze the data without determining the concentration and simply use the absorption to compare results? Is absorbance the same as concentration when talking about chlorophyll?

I’m stuck analyzing the data I obtained from the spectrophotometer. For each sample, I have the absorption against wavelength from the spectrophotometer. What I am planning on doing is to take the $\lambda_{max}$ for each sample and use the absorption from there so that I can utilize Beer’s Law to determine concentration. (Also I want to double check if this law is fit for my investigation)

$\textrm{Absorbance} = \textrm{molar absorptivity} \times \textrm{length of light path} \times \textrm{concentration}$

I know the absorbance and length of light path (length of cuvette), but I am still unsure what molar absorptivity exactly is. How do I determine it? I read that this can usually be found in literature, but I’ve been unable to find it for chlorophyll. Therefore, I was thinking that as molar absorptivity should be a constant (is this true? I have also read that it depends on wavelength), I could leave it as is in the calculations. What I mean by this is:

If molar absorptivity = $\epsilon$, I could leave the concentration as, for example, $1.49/\epsilon$. Would that make sense in a lab report? However, obviously, it would be much better if I could get precise molar concentrations for each sample.

My concern for this method of processing is that I will be ignoring how the wavelength $\lambda_{max}$ varies in every sample. I mentioned this earlier, but if what I read about molar absorptivity varying based on wavelength is true, wouldn’t that affect my concentration calculations? Does that make sense?

In short, if I want to measure chlorophyll degradation (or amount of chlorophyll present), do I need to:

a) Just compare $\lambda_{max}$ (wavelength where absorption peaks) between the different $\mathrm{p}H$ samples

b) Just compare absorption at lambda max between the different $\mathrm{p}H$ samples

c) Compare absorption at one specific wavelength (if so, how do I determine the wavelength if $\lambda_{max}$ differs for each measurement?)

d) Determine concentration from absorption based on $\lambda_{max}$ using Beer’s law, and compare that between the $\mathrm{p}H$ samples?

Answer 1

How to determine chlorophyll content from spectrophotometry data (absorbance vs wavelength)?

You have to consider two pigments, chlorophyll A and chlorophyll B, along with other substances that absorb in the same range. This paper gives extinction coefficients for both pigments, and a recipe to measure at two wavelengths to determine both in a mixture. There are newer papers with extinction coefficents for other solvents that also discuss degradation product, e.g. this one (behind a pay wall).

In short, if I want to measure chlorophyll degradation (or amount of chlorophyll present), do I need to: a) Just compare $\lambda_{max}$ (wavelength where absorption peaks) between the different $\mathrm{pH}$ samples b) Just compare absorption at lambda max between the different $\mathrm{pH}$ samples c) Compare absorption at one specific wavelength (if so, how do I determine the wavelength if $\lambda_{max}$ differs for each measurement?) d) Determine concentration from absorption based on $\lambda_{max}$ using Beer’s law, and compare that between the $\mathrm{pH}$ samples?

I would keep things simple and choose a wavelength and stick with it, using the absorption as an estimate of the total concentration (option c). In the image below, you see a conceptual diagram of the absorption spectra. Your observed spectrum will be some summation of these and the spectra of other absorbing material in your sample. If you measure at about 650 nm, you will capture both A and B. In reporting, you should also show the complete spectrum of your samples to give the reader an idea how different the composition from sample to sample is.

Source: https://ib.bioninja.com.au/standard-level/topic-2-molecular-biology/29-photosynthesis/action-spectrum.html