We want to use phenol pH graphic to measure the pH of mediums before and after inhibitor application to cells. We have phenol absorbence values at 460, 560 and 720 nm wavelength. When we measure the mediums' pH which wavelength must I use?
My choice would be to use a ratio of 2 wavelengths. That would make the measurement of pH independent of the concentration of the indicator (following subtraction of the background absorbance of the medium). Since the 560 and 415 nm absorbance peaks respond in opposite ways to the pH, their ratio would be the most sensitive way to measure the pH. You will need to prepare a standard curve, in which the absorbance peak ratio is plotted on the y-axis and pH is plotted on the x-axis. The pH could then be read off the graph after measuring the ratio of the background-corrected absorbances at those wavelengths of the unknown solution.
I agree that I would start with a ratio measurement. If you have low background absorbance, you may be able to calculate the pH from the ratio and knowledge of the dissociation constant of the due and protons. The math would be similar to that used for florescence ratio determination of calcium as described in the landmark paper link below.
Article A new generation of Ca2+ indicators with greatly improved fl...
The pka of phenol is 9.99. All the pH values shown in the graphic are below this value. If you intend to work within this pH range (where the acidic form of phenol predominates), I suggest to use absorbance values measured at 560 nm which are much more sensitive to pH changes than those measured at 415 nm.
As suggested above, you need to draw calibration curves and work at a constant phenol concentration.
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