The third and fourth phases comprise the elution, i.e. the desorption of the bound molecules, which can be achieved via changing the composition of the elution buffer. The simplest form of elution is achieved via an increase in ionic strength, i.e. in the concentration of the counterions present. Another means of desorption is the change of the pH of the medium. The most effective method is the continuous change of the ionic strength or the pH, i.e. the application of a gradient elution. During gradient elution, molecules with smaller net charges (i.e. the weaker-binding ones) will be the first to leave the column