5. Protein Techniques

What is the order of elution of the following proteins from a cation-exchange chromatography column? 

Net charges of Proteins: Protein A = +1              Protein B = -2                Protein C = -5                Protein D = +3.

In a cation-exchange column at neutral pH, which peptide would elute last?

Mixtures of amino acids can be analyzed by first separating the mixture into its components through ion exchange chromatography. Certain amino acids placed on a cation-exchange resin containing sulfonate groups (—SO₃^-) flow down the column slowly because of two factors that influence their movement: (1) ionic attraction between the sulfonate residues on the column and positively charged functional groups on the amino acids, and (2) hydrophobic interactions between amino acid R-groups and the strongly hydrophobic backbone of the polystyrene resin. For each pair of amino acids listed below, circle the amino acid that is eluted first from the cation-exchange column by a buffer at pH 7.

Give the order of elution of the following peptides when using cation-exchange chromatography at pH 7.2.

Peptide #1: A-D-G-H-E.           Peptide #2: K-L-M-R-A.           Peptide #3: M-D-L-I-V.             Peptide #4: I-L-R-P-M.

Order of Elution: _______________, _______________, _______________, _______________

                                 (1   ^st to elute)                                                                             (Last to elute)