Question

If we have a mixture of Phe, pI 5.5; Thr, pI 6.5; Asp, pI 3; His, pI 7.6 and Lys, pI 9.8, at pH 11 and we load it on anion exchange column and then elute it with a pH gradient of lowering pH, in what order will the amino acids elute?

Answer 1

We know that at isoelectric point the amino acid bears no charge (when pI = pH , net charge =0)

At a pH < pI, : the charge on amino acid = positive

If we start with a particular pH (above 11), where the anion exchange will be positive and all the amino acids are negatively charged (because all have pI < pH), now as we go on decreasing the pH the pI of amino acids will start exceeding the pH and hence it will be eluted

So the sequence will be

Lys pI = 9.8

His : pI 7.6

Thr, pI = 6.5

Phe, pI = 5.5

And finally Asp , pI = 3