HEPES - the when, why, & how of using this pH buffer

HEPES stands for N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. People can’t agree on how to pronounce it, but many do agree that it is often a “Good” buffer to use to stabilize the pH of a biochemical solutions!
blog: bit.ly/heapsofhepes
Much more on buffers in other posts - especially this one: bit.ly/phbuffers ; RUclips: • pH buffers - an overview & • Choosing and preparing...
But basically they’re pH-stabilizers. pH is a measure of free proton availability. It’s an inverse log scale, so the more protons are available for molecules to snatch up, the lower the pH (and we say this is more acidic). Conversely, the fewer protons are available (the more basic or alkaline the solution), the higher the pH.
“Bases” are (at least in one definition) molecules that snatch up protons. And doing so turns them into acids! (Molecules that donate protons). We refer to these as the conjugate acid and conjugate base form of a molecule and molecules can go back and forth between them. But they might not want to…
Some molecules are really reluctant to have a proton. We call these strong acids. And some molecules are really reluctant to give up a proton. We call these strong bases.
But there are also molecules that are pretty happy either way. We call these weak acids/bases and they can make great buffers (at least near their pKa). The pKa is the value at which half of the copies of the molecule are in the acid form (protonated) and half are in the base form (deprotonated). Above that, there aren’t enough protons available to satisfy it, so most are deprotonated. And below that, protons are abundant so most will be protonated. But near the pKa, there will be lots of both forms available. And thus you will have both molecules that can sop up added protons and molecules that can donate protons if protons get used up. As a consequence, around the pKa, the pH will stay! And these molecules thus make good buffering agents around their pKa (but not away from it)
Different buffer molecules have different pKa values so can be used to buffer solutions at different pH values. In biochemistry and biology we usually want to work in pretty “physiological” (bodily realistic) conditions, which requires a pH of ~7.4. There are a variety of buffers that fit the bill - including HEPES, which has a pKa of ~7.5 (depending a little - but not a lot - on the temperature).
You see, one great thing about HEPES compared to another common buffer, Tris, is that HEPES is influenced less by temperature (though it too will drift upwards. 20mM HEPES has a pKa of ~7.55 @20°C, which creeps downwards at a rate of -0.140/10°C. Tris, on the other hand, has a pKa of ~8.30 @20°C, which creeps downwards at a rate of -0.310/10°C.
Temperature Dependence of pH for Commonly Used Buffers, Promega, referencing Good,N.E.(1986)Biochemistry 5,467. www.promega.com/-/media/files...
But Tris has a higher buffering range so if you need something closer to a pH of 8, that might be a good option.
HEPES can also be useful in cell culture as an additive on top of conventional bicarbonate buffer systems. Much more on those in another post: blog: bit.ly/phenol_red_pH ; RUclips: • Phenol red and pH buff...  
But basically they rely of carbon dioxide in the co2-controlled incubator to provide some protons when it dissolves. So if you take your stuff out of the incubator, the lower CO₂ concentrations will cause the pH to rise. But if you have HEPES in there, it can take over for the co2 and keep things steady. You don’t want to use too much HEPES though because it can cause problems at high concentrations and potentially affect some cell stuff. And you want to keep it protected from light because toxic oxidation products can form.
There are also a bunch of other things to consider when choosing a buffer. One is whether they bind to metals (act as chelates). HEPES “doesn’t” which is good if you have metal-dependent proteins, reactions, etc. Much more in that buffer post I link to at the top.
HEPES is on the list of “Good’s buffers” - and no, that’s not a typo. Thats the name for 20 buffers a scientist named Norman Good and his colleagues found had qualities that made them useful for biology and biochemistry (low toxicity, good solubility, useful buffering ranges, etc.). They published their findings in 3 papers spanning from 1966 to 1980.
Here’s the first one (in which HEPES made the cut, by the way!)
Good, N. E., Winget, G. D., Winter, W., Connolly, T. N., Izawa, S., & Singh, R. M. (1966). Hydrogen ion buffers for biological research. Biochemistry, 5(2), 467-477. doi.org/10.1021/bi00866a011
And this article has some nice info: “The origin of Good’s buffers” by Robyn Bennis, Gate Scientific gatescientific.com/technique-...
finished in comments

[@thebumblingbiochemist]

As for making HEPES buffers, it’s common to make a 1M stock solution. To do so you usually dissolve the free acid form (238.3 g/L) in a partial volume and then take away protons by adding in a strong base (in the form of NaOH or KOH depending on which of these counterions you want hanging around). It will take a lot, so it can be good to start with pellets of the base and then switch to a concentrated solution to do the final adjustments.
Much more on making buffers here: ruclips.net/video/jJBbCj3Kz7c/видео.html
Further reading:
Ferreira, C., Sousa-Pinto, I., Soares, E. V., Soares, H. M. V. M. (2015). (Un)suitability Of the Use Of Ph Buffers In Biological, Biochemical And Environmental Studies And Their Interaction With Metal Ions - A Review. RSC Adv., 39(5), 30989-31003. doi.org/10.1039/c4ra15453c
The components of the cell culture medium: HEPES, December 15, 2021, CellCulture, cellculture.altervista.org/the-components-of-the-cell-culture-medium-hepes/?doing_wp_cron=1686487460.1280500888824462890625
Effect of temperature on pH: Häring M, Pérez-Madrigal MM, Kühbeck D, Pettignano A, Quignard F, Díaz DD. DNA-Catalyzed Henry Reaction in Pure Water and the Striking Influence of Organic Buffer Systems. Molecules. 2015; 20(3):4136-4147. doi.org/10.3390/molecules20034136
   
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com

[@DomenikStuhlmacher]

Really good video! Thank you for your effort!

[@thebumblingbiochemist]

Thank you!