Effect of pH on Elution Pattern (SP-825)

Using an eluent at pH of 7.0 or 8.0, five types of proteins are well separated using IEC SP-825 (a column for strong cation exchange chromatography). The retention time of ribonuclease A increases with decreasing pH value and, at pH of less than 6.0, ribonuclease A elutes after alpha-chymotrypsinogen A. At pH 6.0, the separation of ribonuclease A from alpha-chymotrypsinogen A is insufficient. At pH 4.0 or 5.0, myoglobin does not elute sharply.

Sample :
1. Myoglobin, 2. Ribonuclease A, 3. alpha Chymotrypsinogen A, 4. Cytochrome c, 5. Lysozyme

Column    : Shodex IEC SP-825 (8.0mmID*75mm)
Eluent    : (A); 20mM Sodium formate buffer(pH4.0)
                 20mM Sodium malonate buffer(pH5.0 and 6.0)
                 20mM Sodium phosphate buffer(pH7.0)
                 20mM HEPES(pH8.0)
            (B); (A) + 0.5M NaCl
            Linear gradient: 0min to 20min, (A) to (B)
Flow rate : 1.0mL/min