Question

Question 5.A mixture contains Gu, His, Pro, Lys and need to be separated using Dowex 50 cation exchange, prepared in a loading buffer at pH 2.0 : the following buffers are available :- -Amino acid mixture in 0.04 M citrate buffer, at pH 2.0. -0.04 M citrate buffer at 4.5 -0.04 M citrate buffer at 8.0 -0.04 M citrate buffer at 10.5 Assume these amino acids elute only at pH above their perspective pl. Support your answers with clear explanations. a) Identify the order of elution b) Identify the amino acids that may eluted together if no complete elution performed.

Answer 1

CATION exchange chromatography has ion exchange resin contain negative charge group where positive charge molecules binds.

If pH < pI means the molecule will have a positive charge.

If pH > pI means the molecule will have a negative charge.

The buffer starts from pH = 2. All the amino acids pI are greater than 2 so, they all are positive charge.

1. At buffer pH 4.5 > pI 3.08 Glu. So Glu become negative charge it will 1st elutes.

2. At buffer pH 8.5 > pI 6.30 Pro, 7.64 His. So Pro, His become negative charge it will elute second together.

3. At buffer pH 10.5 > pI 9.47 Lys. So Lys become negative charge it will elute third.

B. Pro, His elutes together.