Question

You have a mixture of the proteins listed below.

Protein	pI	Molecular Weight (kDa)
A	3.1	265
B	6.9	93
C	10.3	96
D	7.1	43
E	8.6	189

1. You load the protein mix onto a cation exchange column at pH 5. Next, you apply a "washing" step by passing through buffer at pH 5. Finally, for your elution step, you apply a pH gradient starting from pH 2.0 to pH 13.0. (A gradient buffer system allows you to gradually and continuously change the pH of your mobile phase starting from pH 2 up to pH 13).

Indicate the order in which proteins will exit the column during the elution step. Explain your answer.

2. You load this same mixture onto a size exclusion column. Please indicate the order of elution. Explain your answer.

3. If your protein of interest is protein B, would using anion exchange or size exclusion be completely successful at separating it from the other proteins? Yes or NO. Explain your answer.  

Answer 1

In cation exchange column, the exchangable ions are cationic, the more the positive charge density on the cation, the more time it retains in the column.

1. Protein mixture at pH-5 would have only protein A in anionic form as its pI is less than the pH applied. So protein A will be eluted first from the column when washing is applied, and other proteins will remain attached to the resin column. Upon applying elution step, with pH gradiant starting from 2 to 13, the protein with lowest pI among B to E will elute first and the protein with highest pI will be eluted at last because the protein having highest pI will be having positive charge on it till the pH of solution crosses its pI. So the orger of elution with incrrasing time is as follows--

B then D then E then C.

2. In size exclusion crhromatography, the column is filled with beads of specific pore size. These are called molecular sieve. The smaller the protein molecule ,the longer it permeates the pores and hence longer the time it will take to elute from column. The molecules having bigger in size doesnot fit the pore size and hence elutes first, so the order of elution of protein with time will be--

A then E then C then B then D.

3.as molecular weight of B and C are quite similar so the , size exclusion will not be able to separate protein B to a rich quality even if separated from other proteins , rather there is a large difference in their pI value of protein B and C. In anion exchange the column or the stationary phase is positively charged and the exchangable ions are anions, i.e. the protien with greater negative charge density will retain to the column for longer time. But the proten B and D having the comparable pI values, the anion exchange technique will also be failed to separate it effectively