I'm wondering that how most authors are saying and plotting this equation. I practically applied this but getting a line parallel to [dna]. Uv titration graph was same as others. Benesi plot is suitable but this wolff-shimmer, if i take total dna concentration never got a plot like many published. Can you or any other clarify me (may i wrong). Taking an overview of dna binding with drug/ligand, addition of dna will increase absorbance (some cases decrease but ignore this case). As free absorbance is constant, so their differences as well as extinction coefficient will increase upon gradually increasing dna concentration. So the plot of (Ea-Ef) vs [dna] will be linear starting from origin. But now if we change y axis as [dna]/(Ea-Ef) then how the plot looks same as before?? I think the [dna] in the plot are two different like total and unreacted.