Question

How would you use ion exchange chromatography to seperate a mixture of alanine, glutamic acid, and lysine if you wished to maintain the pH at 7.5 and use only a single ion exchange column ( either an anionic or cationic exchanger). Describe the expected order of elution under the conditions which you propose.

Answer 1

The ion exchange chromatography is a process where the ions and the polar molecules are separated based on their ion affinity towards the ion exchanger and helps in separation of wide range of charged molecules. It helps in separating proteins, amino acids and nucleotides. There are immobilized charges groups that are used as stationary phase and as the mixture of the amino acid are moved through it, based on the reversible adsorption to the charged solute molecules, it is separated. The pI of alanine, glutamic acid and lysine at 25 degree C is 6.11, 3.08 and 9.47 and hence the glutamic acid has highest and the lysine has the lowest. At pH 7, the glutamic acid is negatively charged and the arginine and lysine is positively charged. In this case, it is important to establish and set up a cationic exchanger as it has more affinity towards the positively charged ions. This will help in attracting the positive amino acids which are alanine and lysine.

In case of the elution, the first be eluted is the glutamic acid as the stationary ion exchange phase that will be used in a cationic and not anionic. Since the glutamic acid cannot bind to the ion exchange, it will be eluted first. This will be followed by the elution of the alanine which has the pI of 6.11 that is less than lysine which has a pI of 9.47 which has the maximum attraction and will be eluted last.