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Many molecular biology protocols advice against sterilizing HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) solutions by autoclave.

Supposedly, HEPES is heat labile, and the high temperatures used for sterilization somehow impair the function of the buffer, or perhaps generate unwanted chemical species (this is speculation on my part; I've often seen the advice to not autoclave HEPES, but never seen anything about why not). Typically autoclaves heat solutions to 121C and increase pressure to slightly above 2 atm, which is about a degree short of the boiling point of water. This environment is maintained for 20 minutes in order to destroy biological contaminants.

I have seen this claim often, but had trouble locating an actual source or reasoning for why HEPES should not be autoclaved. Has there been a publication that actually tried to heat a HEPES buffer to autoclave temperatures (121C) and see what happens? What exactly is the consequence, especially with regard to the biological uses of HEPES (ie. pH buffer)?

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From here:

Some buffers (e.g., MOPS and HEPES) cannot be autoclaved because they degrade upon heating.

and from here:

[HEPES] Solutions may be autoclaved under standard conditions.

I guess nobody knows the answer.

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  • $\begingroup$ So there isn't even a consensus about it. Interesting! Unfortunately the source for the second claim, Medzon and Gedies 1971, is paywalled and beyond my reach, so I'm a little stuck there. $\endgroup$ – Superbest Sep 4 '14 at 5:04
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    $\begingroup$ Sorry, I can't access it either. It's likely that they autoclaved the HEPES solution and found out that it was still usable. It might be that some autoclaves have a slightly higher working temperature (like 125°C), so the HEPES is more likely to degrade in those autoclaves. $\endgroup$ – LDC3 Sep 4 '14 at 5:25
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Today I tested a set of competent E. coli cells that were prepared using a HEPES-based calcium chloride buffer solution. The solution was autoclaved as I described in the answer.

I obtained a fair transformation efficiency from these cells, and viability was also not perturbed noticeably. There are several confounds in the preparation of my solution that could affect the transformation efficiency, but in conclusion, autoclaving the HEPES has not abrogated its ability to make cells chemically competent.

I will speculate further and say that autoclaving HEPES may degrade some of the HEPES molecules or produce reactions and generate unexpected subspecies - but both of these will be generated in small, insignificant amounts which should not affect most biological applications.

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I've read the paper of Medzon & Gedies. They autoclaved HEPES medium and see no statistical difference between it and bicarbonate solution in cell culturing. There is nothing said about impossibility of HEPES autoclaving.

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