Question

Explain how to use an ion exchange column containing carboxymethyl resin to separate a protein mixture containing insulin (pl = 5.4), hemoglobin (pl = 7.1) and cytochrome c (pl = 10.6).

Answer 1

Carboxymethyl is a negatively charged molecules and thus is used as cation exchanger because it attract the positive charge.

So, here , the mixture contains insulin, hemoglobin and cytochrome C.

isoelectric pH is the point of pH at which the molecules remains in zwitterion mRNAs doest have net charge .

As, we know pH more than isoelectric pH gives molecules net negative charge because the H+ concentration of the solution becomes low when pH is increased and that why molecules emit H+ and get negative charge.

So if you applied a buffer in chromatography which has pH more than 5.4 and less than 7.1 like 6 then, insulin will have net negative charge and hemoglobin will have negative charge and CYTOCHROME C will also have positive charge so , insulin will not bind to the resin and it will be eluted when wash buffer will be applied whose pH will be more than 5.4.

Now, in resin cytochrome cand hemoglobin is attached and you have to isolated them.

Now you have to make a wash buffer whose pH will be more than 7.1 and less than 10.6 like 9 ,in this pH the HEMOGLOBIN will have. negative charge but the CYTOCHROME c wi have positive charge and thus hemoglobin will be eluted and cytochrome C will be attached within resin.

Now if you applied wash buffer of pH more than 1.6 then cytochrome C will developed negative charge and thus it will also be eluted.

Thus, 3 can be separated.

Thanks a lot.

Good luck