Question

1. You want to separate two proteins, Protein A and Protein B, using ion exchange chromatography. Protein A has a pI of 4, and Protein B has a pI of 8.

a) To get optimal separation of your proteins, what pH should your elution buffer be? ____________

b) At your chosen pH, would the charge on Protein A be ¨ positive, ¨ negative, or ¨ neutral?

c) What about the charge on Protein B at the chosen pH? It is ¨ positive   ¨ negative   ¨ neutral

d) If you used anion exchange chromatography which protein elutes first?

Answer 1

a) The ion exchange chromatography is a separation method where the analytes are separated according to their charge. This chromatography is formed by a stationary phase that has ionic functional groups in its surface and by a mobile phase that is a buffer. In this way, the analytes loaded are retained differentially by the stationary phase, so they have different retention times.

The isoelectric point (pI) of a protein is a pH where the protein has a net charge equal to 0. So,

If the pH is higher that the pI, the net charge of the protein will be negative.

If the pH is less that the pI, the net charge of the protein will be positive.

So that, if we have a Protein A with a pI of 4, and a Protein B with a pI of 8, we can choose a pH that is found between the isoelectric points of both proteins. So that, both proteins will have different charges and can be separated by this type of chromatography. This pH can be 6.

b) The chosen pH is higher that the pI of Protein A. So that, its net charge is negative.

c) The chosen pH is less that the pI of Protein B. So that, its net charge is positive.

d) In an anion exchange chromatography the stationary phase has ionic functional groups with positive charge so that, the protein A with negative charge will be retained with greater force than protein B. So, the protein B will come out of the column first and after that, the protein A.