Can one determine absorption(extinction) coefficients using the CLARiTY?
“Yes. And this is true whether the sample has turbidity or not.”
The following was penned by Prof Robert C. Blake II, the first person to purchase a
CLARiTY UV/Vis spectrophotometer (2009). Bob has pivotal publications using the
CLARiTY, including a 16-page chapter in Advances in Microbial Physiology, Volume 76
(2020). This answer written in February 2023:
When I was first starting to explore using the CLARiTY, years ago, I VERIFIED values of well-known absorption coefficients using well-known things like ferricyanide, cytochrome c and myoglobin both in the presence and the absence of purposeful light-scattering suspensions. In all cases, when I did a titration of the absorbance values at different concentrations of the colored materials, a nonlinear, concave-down curve was obtained.
However, when I corrected the raw absorbance data to absorbance values per cm using methods attributable to either Fry or Javorfi, linear curves of absorbance versus concentration were obtained that permitted the true absorption coefficient to be calculated using Beer’s Law.
More recently, I have been determining the unknown absorption coefficients for colored, intact microorganisms using the same approach: titration of raw absorbance values as a function of different concentrations of the intact microorganisms; followed by correction of the raw absorbances to absorbance values per cm using the Fry method. These titration curves are biphasic. The first phase at any wavelength is a rapid decrease in absorbance (to negative absorbance values!) that bottoms out at a fixed negative value that is independent of the identity or color of the microorganism. The second phase is then a linear curve of absorbance as a function of the concentration of suspended microorganisms, from which a Beer’s Law absorption coefficient is readily extracted.
I haven’t published any of these results, although it is my intention to do so. I am encouraged that you think that these observations might be useful. Anyway, I would be happy to argue with anyone who wanted to pursue such a discussion further.