A series of samples are prepared with all the same receptor (R) concentration but in which the ligand (L) concentration is titrated. After equilibrium is reached each sample is measured by flowing it through the column (Figure 2).
For 1:1 reversible binding Equilibrium Kd is defined as
(1)     K≡k/k =R*L/RL
the binding is reversible so conservation of mass can be written as
(2)     R = R+RL
(3)     L = L +RL
Where:
K = equilibrium dissociation constant
k = forward rate constant
k = reverse rate constant
R = free receptor site concentration at equilibrium
L = free ligand site concentration at equilibrium
RL = concentration of complex at equilibrium
R= total concentration of receptors
L = total concentration of ligand
A simple equation relating the free fraction of R (=R/R) to the K and L is then fit to the measured data to find the K of the interaction.