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Effects 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 α-chymotrypsinogen A. At pH 6.0, the separation of ribonuclease A from α-chymotrypsinogen A is insufficient. At pH 4.0 or 5.0, myoglobin does not elute sharply.

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

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

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