I'm very confused on how to calculate the [A-]/[HA] value, given the fact that my dilution series uses buffers at various pH values with the same indicator (supplied at the same concentration to each diluent).
For example, I have been told that for the diluent composed of acidic buffer (pH=3, supplied at concentration of 0.2M, volume of 800µL) and indicator (concentration of 0.20µM, volume of 400µL), that the indicator is 100% protonated. As well, for the diluent composed of basic buffer (pH=9, supplied at concentration of 0.2M, volume of 800µL) and indicator (concentration of 0.20µM, volume of 400µL), the indicator is 100% deprotonated. I'm quite unsure what to even consider as the [A-] and [HA] (and or [B] and [BH+]) terms, since I'm working with both types of buffers but the same indicator.
As well, I have measured the absorbance value (using the same wavelength=480nm each measurement) across the dilution series (pH ranging from 3-9, using different buffers with varying pH values).
I'm just quite confused how to determine the pKa of my indicator, as well as the [A-]/[HA] values, based on the absorbance value at that given pH (as well as relating the differing concentrations of indicator and buffer). I would really appreciate if anyone could provide clarity on how to re-arrange the Henderson-Hasselbalch equation, and Beer-Lambert law, to determine these values and calculate the pKa, in this context.
Thank you, in advance.
