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Hepes Tris pH 6 buffer

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#1 PaulF



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Posted 03 June 2011 - 03:11 AM

Can anyone help with this?
I need to prepare a buffer which has been described in several papers as "20mM Hepes/Tris (pH Hepes/Tris 6.0)"
I'm not sure what the 20mM refers to (is it the Hepes or Tris) and how to get it to pH 6.0

Any help would be much appreciated.


#2 phage434



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Posted 03 June 2011 - 05:16 AM

Tris is a base, and HEPES is an acid, so this buffer is a mixture of the two nearly neutralizing each other. There is ambiguity, but I would assume the 20 mM is the final concentration of the two components (though this is over-specified, since they also specify pH). An alternative interpretation, and easier to make, would be 20 mM of either one. The pKa of Tris is about 8.05 and of HEPES about 7.55, so neither of these is a very good buffer at pH 6.0. The original experimenters would have been better off using PIPES (pKa 6.7) instead of HEPES.

Roughly speaking, make 40 mM solutons of Tris base and HEPES (may not go into solution) and titrate to pH 6.0. Given the solubility of HEPES, you might want to start with solid HEPES in the correct amount and add 40 mM Tris-base until it is dissolved, then titrate to pH 6.0.

#3 PaulF



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Posted 03 June 2011 - 06:04 AM

Many thanks. I thought this was probably the way to do it but the pH threw me a little.
I'll give it a go.

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