Dear all,
I am making a 10mM KHPO4 buffer (pH 7) for protein purification.
I need to add 350mM Imidazole to it for the purpose of protein elution, but after the addition of Imidazole pH jumps to 10.
So how can I adjust the pH to 7?
Shall I use phosphoric acid or hydrochloric acid?
Is it recommended if I make 1M stock of Imidazole and adjust its pH to 7 using HCl and then I add this to KHPO4 buffer?
Please suggest.
Thanks
Dear Deepak Deepak ,
Imidazole acts as a base with a pKb = 7.0 and consequently a pKa of round 7.0 as well (and a weak acid pKa = 14.9) and it has been reported that once you dissolve it in water you get a pH 10.5 for 38 g/L at 20 °C:
https://pubchem.ncbi.nlm.nih.gov/compound/Imidazole#section=Dissociation-Constants
So with a pKa value of approximately 7.0 and at a pH of round 7.0 both the acid and base forms are present. Therefor it has been reported that Imidazole can be used to prepare buffers in the pH range of 6.2-7.8 at 25 °C.
In trying to answer your question, according to a well-described protocol (especially pages 20 and further):
https://www.gelifesciences.co.jp/tech_support/manual/pdf/71502043.pdf
the best way is to adjust the imidazole to pH 7 first (just using HCl).
Best regards.
Hi Deepak,
your buffer capacity of your 10 mM KHPO4 is by far (35 times) exceeded!
- adjust molarity of the KHPO4 buffer also to 350 mM - maybe not desirable
- or - as you suggested - neutralize imidazole and adjust it with acid to pH 7 in advance. Which acid you use depends mainly on which ions you want to tolarate
Dear Deepak Deepak ,
Imidazole acts as a base with a pKb = 7.0 and consequently a pKa of round 7.0 as well (and a weak acid pKa = 14.9) and it has been reported that once you dissolve it in water you get a pH 10.5 for 38 g/L at 20 °C:
https://pubchem.ncbi.nlm.nih.gov/compound/Imidazole#section=Dissociation-Constants
So with a pKa value of approximately 7.0 and at a pH of round 7.0 both the acid and base forms are present. Therefor it has been reported that Imidazole can be used to prepare buffers in the pH range of 6.2-7.8 at 25 °C.
In trying to answer your question, according to a well-described protocol (especially pages 20 and further):
https://www.gelifesciences.co.jp/tech_support/manual/pdf/71502043.pdf
the best way is to adjust the imidazole to pH 7 first (just using HCl).
Best regards.
Just titrate the imidazole with HCl and omit the phosphate buffer - the imidazole will act as a buffer in this case.
As a side note - you can make 1M imidazole stock, titrate it with HCl to ~7-7.4 and use this to mix your buffers for protein purification.
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