The c value is defined as c = [M ] 0 K d with [M ] 0 being the concentration of the titrand and should ideally be between 1 and 1000 for reliable determination of the dissociation constant. This means that there is both an upper limit and a lower limit for the determination of the thermodynamic parameters. Low c value titration is feasible and has been shown to give valid K d values, but it requires a lot of both fragment and protein and errors regarding ΔH are likely. Displacement titrations have been carried out for weak binding fragments as well as for ligands with a high affinity in the one-digit nanomolar range. The displacement experiment for compounds showing low affinity towards the target requires the presence of a high affinity ligand that binds to the same binding site as the weak one. Prior to the titration, the protein is incubated with the fragment and in the following ITC experiment, the strong ligand displaces the weak binder from the binding site. The displacement titration as well as the one with the strong ligand yield affinities (K observed for the displacement titration and K reference for the titration with just the ligand) that can be used to calculate the K fragment of the weakly binding molecule (equation 2).