Dear all,
I noticed that the pH rises when I dilute a 2 M stock solution of imidazole (pH 8.0) into a 0,2 M phosphate buffer (pH 8.0) to 500 mM imidazole (His-Column elution buffer). The buffer also contained a solution of amino acids (also pH 8.0).
pH went from 8.0 to 8.3 - why does it happen? Is the buffering capacity of phosphate exhausted?
Is this a known effect and is the only possibility to prevent this by doing pH-adjustment after dilution?
thanks for any advice,
Best,
Peter
Hi there,
At pH8 imidazole is totally deprotonated and acts as a weak base when mixed with phosphate buffer (which has not a strong buffer effect at pH8). Furthermore imidazole is more concentrated than the buffer system at the end. So imidazole is binding proton freed by acid function of phosphates.
An additional factor may be the activity (in physical chemistry terms) of the imidazole. The activity of a 2M stock solution may be less than 10 times the activity of an 0.2M solution. As a result, when the 2M stock solution at pH 8.0 is diluted 10-fold, the diluted solution has a higher pH (because imidazole is basic), and the phosphate buffer is unable to buffer it completely.
I also thought about activity, but did not expect that it could have such a pronounced efect. i think that solves the case, together with the low capacity - thank you two!
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