Change of pH of Phosphate buffer with Imidazole - (Aug/03/2010 )
I am preparing a set of phosphate buffers for my protein purification. I have the stock phosphate buffer 500 mM pH 7.8. I dilute to make 20 mM buffer. However, If I add NaCl (500 mM) and Imidazole (20, 50 and 500 mM) the pH changes considerably. In that case, how to make the final pH to 7.8? Can I add HCl or one of the phosphate solutions to adjust the pH? Will it not affect the ionic strength of the final buffer? I want all the buffers at same pH and ionic strength as I am going to use them for gradient wash/elution.
Many thanks in advance.
the imidazole is causing the change in pH. imidazole is a buffer (pK 7.0). you can preadjust a stock of imidazole or adjust in the solution (don't use phosphate, you want that to remain constant). any change in ionic strength should be inconsequential.