You come to the lab one day and find that the highschool student who is helping

Question

You come to the lab one day and find that the highschool student who is helping you has mixed all these samples together. As you supress your desire to throttle him/her you realize that you can use anion exchange chromatography to seperate out the peptide mixture. In anion exchnage chromatography the resin in the column is positively charged. Solutes that are net negstively charged will be retained on the column while net neutral or net positively charged solutes will pass through the column unbound. The negatively charged solutes bound to the column can then be removed using a salt gradient. The order in which the bound solutes are eluted will depend on the charge on each solute at the ph being used. Solutes with a few negative net charges will be bound to the column weakly and elute firts, while solutes with more net negative charge will be bound to the resin stronger and will elute later.

Question:

If you added your peptide mixture to an anion exchange column at ph 9, what would you expect to flow through(not be retained) and what would you expect to stick to the column? Why?

Explanation / Answer

A peptide mixture may contain peptides made up of various amino acids with various side groups(that may be either acidic or basic) apart from the terminal -NH3 and -COOH groups of the peptide. So at pH=9, which is basic condition, the acidic side groups may get deprotonated and get negatively charged.

1) So those peptides with amino acids having acidic side groups(eg aspartic acid, glutamic acid) will get deprotonated, provided their pka <9 .Such peptides would be retained by the chromatographic column.

2) the net negative charge of the peptide must be negative to be retained.

3) the peptides with amino acids having basic side groups-NH2 (eg arginine) would not be effected and stay neutral. If the net charge of the peptide (net charge of all terminal and side groups) is positive ,it will be eluted by the resin.

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