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
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
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