Question

1. An anion-exchange resin can be used instead of the cation-exchange resin in this experiment to separate the three amino acids in the known mixture. What changes to experimental protocol are required to achieve complete resolution? Support your response with an explanation. (HINT: consider pH).

2. You have been provided a mixture containing four amino acids (Glu, His, Pro and Lys) to be separated by ion-exchange chromatography using a Dowex 50 cation-exchange, prepared in a loading buffer at pH 2.0. The following solutions are made available:

3. The amino acid mixture in 0.04M citrate buffer at pH 2.0.

4. 0.04M citrate buffer, pH 4.5

5. 0.04M citrate buffer, pH 8.0

6. 0.04M citrate buffer, pH 10.5

   Is complete resolution of all amino acids achieved if the buffers are allowed to elute through the column sequentially from lowest to highest pH? Identify the order of elution and identify any amino acids that may come off the column together if complete resolution is not achieved. Support your response with an explanation.
   Assuming the amino acids in the mixture will elute from the column ONLY at a solution pH that is above their respective pI.

Answer 1

Solution:

Important notes:

1. In ion-exchange chromatograph, the molecules bind to oppsitely charged sites in the stationary phase when they passes through the chromatographic column.
2. Isoelectric point (pI) is the pH at which an amino acid has anet charge of zero. That means zwitterion form is dominant.

Effect of pH on -NH2 and -COOH group of the protien is tabulated below.

Amino acid	pI	Ions available at pI	pH = 2.0	pH = 4.5	pH = 8.0	pH = 10.5
Glu	3.08	-NH3+ ; -COO-	-NH3+ ; -COOH	-NH2 ; -COO-	----------	----------
Pro	6.30	-NH3+ ; -COO-	-NH3+ ; -COOH	-NH3+ ; -COOH	-NH2 ; -COO-	----------
His	7.64	-NH3+ ; -COO-	-NH3+ ; -COOH	-NH3+ ; -COOH	-NH2 ; -COO-	----------
Lys	9.47	-NH3+ ; -COO-	-NH3+ ; -COOH	-NH3+ ; -COOH	-NH3+ ; -COOH	-NH2 ; -COO-

Initially at pH = 2.0, all the 4 aminoacids are in the form of -NH3+ ; -COOH. Hence all of them will be bonded with the stationary phase. Glu will be eluted from the column after the pH of solution reached to 4.5 beacuse -NH3+converted to NH2 and - COOH to -COO-. At pH 8.0, Pro and His will elute together. Finally, Lys will be eluted when the solution pH reaches to10.5.

Hence using the conditions mentioned in the question, we shall separate Glu (First eluting) & Lys (Last eluting) from the mixture. Pro & His (Second eluting)will be eluted as mixture.