Hi there,
in an experiment I used a Cary-60 UV-Vis Spectrophotometer (Agilent Technologies) to collect UV-Vis reflectance spectra of cyanobacteria (~5µm cell diameter) on cellulose nitrate filters. I divided the cell spectra by the spectra of the filters without cells as a reference. Then I applied the Kubelka-Munk function F(R) on this reflectance spectra in order to obtain absorbance spectra.
Now, do the resulting values from F(R) in numbers really represent absorbance, so that it makes sense to include them in the axes of my graphs for my thesis? My values of reflectance were between 0.2 and 1 and the resulting F(R) values are between 0 and 1.4.
Here (http://www.uco.es/organiza/departamentos/decraf/pdf-edaf/DRS08.pdf) I have read that for Kubelka-Munk to apply the grain size should be large in comparison to the used wavelength (which is the case I guess, assuming I can use cell size as particle size?) and significant deviations occut at R
Dear Max, if you measure diffuse reflectance you must be aware of the fact that there is no analytical way to solve the inverse problem and gain the optical constants of your material(s) from the spectra. Applying Kubelka-Munk is an approximative way of solving this problem, but the absorbance you obtain by this procedure is certainly not the same as the one gained by a transmittance experiment. On the other hand, absorbance is nothing but an artificial quantity that somehow relies on the assumption that the Beer-Lambert law is applicable which is not a even a given in transmission experiments. E.g. even a thin homogeneous layer on an index matched substrate will in general not follow the Beer-Lambert law. If you want to know more about the Beer-Lambert law, here is a review about it: Article The Bouguer-Beer-Lambert Law: Shining Light on the Obscure
Concerning diffuse reflectance the reference about how to treat such spectra for me is still a book from the 60ies of the past century: https://www.springer.com/de/book/9783662282700
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