Question

You are trying to separate a mixture of Glutamate, Asp, and Leu. You run the mixture over an anion exchange column (buffer pH = 6.0). In what order should the amino acids elute?

Hint: Consider the side chain pKas

Answer 1

when pH lower than pKa: group is protonated

when pH higher than pKa: group is deprotonated

Glu: Pk(a-COOH): 2.16 => deprot. negative                                      

       Pk(g-COOH): 4.32 => deprot., negative

      Pk(NH2): 9.96 => protonated, positive

    overall charge: negative

Asp: Pk(a-COOH): 1.99 => deprot. negative                                      

       Pk(g-COOH): 3.90 => deprot., negative

      Pk(NH2): 9.9 => protonated, positive

    overall charge: negative

Leu:Pk(COOH): 2.33 => deprot. negative                

      Pk(NH2): 9.74 => protonated, positive

    overall charge: neutral

anion exchange means the solid phase is positive charged. negative charged ion will stick to the solid phase.

First the neutral leu will elute, then the glu, because the pka value is bigger, that means it is less deprotonated than Asp. but I am not sure if a very good seperation of Asp and glu can be obtained, due to the very close pka values