Solvent A

For ion exchange chromatography Solvent A is usually a 0.02 - 0.1 M buffer solution of the desired pH, containing little or no additional salt. It is the lower limit and column equilibration buffer for the gradient. Solvent A is designed so that the protein of interest will bind to the stationary phase during loading and washing, then will elute when a salt gradient is applied.

It is sometimes advantageous to add salt to Solvent A, the lower limit buffer, so that weakly charged proteins will not bind to the stationary phase at all. Compare the elution of the four proteins in the following separations. You will see that by increasing the salt concentration in Solvent A, one of the proteins does not bind to the stationary phase and elutes immediately after loading.

Stationary Phase CM-Sephadex CM-Sephadex
Solvent A 20 mM Sodium Acetate, pH 8.4 containing 0.0 M NaCl 20 mM Sodium Acetate, pH 8.4 containing 0.12 M NaCl
Solvent B 20 mM Sodium Acetate, pH 8.4 containing 0.2 M NaCl 20 mM Sodium Acetate, pH 8.4 containing 0.2 M NaCl

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