buffer chemists, please hope me!

June 20, 2007 6:05 PM

ChemistryFilter: I want to buffer a solution to pH 4.5. The catch -- no carbon compounds or phosphate allowed. Any suggestions for low- to mid-range pH buffers that don't contain C or PO₄^3-?

We are developing an enzyme assay that must use a buffer that is not carbon-based, for various reasons. Normally I would use a phosphate buffer for this pH range, but phosphate inhibits some of the enzymes we're measuring, so no phosphate is allowed either. A borate buffer won't work because pH 4.5 is too far below the pKa of boric acid (about 9). Every other inorganic acid with a pKa in the correct range that I've found (hydrofluoric, hypochlorous, arsenic, chromic, selenious, tellurous, nitrous) is either fairly toxic, unstable, or both. Any other suggestions?

This is not a homework question! I wish it was -- answers would be easy to find if that was the case.

posted by harkin banks to Science & Nature (13 answers total)

My dad is a chemical engineer, and I asked him your question. The first thing he said was "that's going to be almost impossible." Then, he said that if you play around with different sulfur compounds, you may have luck. He also suggested asking a college professor -- there was one he recommended from his days at Case (Professor Olah), but he said that he's almost definitely retired by now.

IANAChemist, but maybe something I wrote will help....
posted by fvox13 at 6:35 PM on June 20, 2007

I scanned, during grad school, a page from the Biochemistry CRC that lists all the "Goode" buffers. Nothing in there fits your bill. However, if you haven't already, you should look in the Chemistry CRC and the biochemistry CRC at the lists of common buffers and pH ranges for those buffers. The CRC books are pretty useful, and if you're university affiliated, you may have online text access.

Good Luck.
posted by u2604ab at 6:41 PM on June 20, 2007

Response by poster: u2604ab: I've got the chemistry CRC here on my desk, but to be quite honest I didn't know there was a biochemistry-specific CRC. I'll check that out.
posted by harkin banks at 6:46 PM on June 20, 2007

I put the question to my former-chemist husband, and he replied that he thinks you're "SOL." When pressed to elaborate, he says:

"I think the other posters covered it. Without using organic compounds there's just not enough options (and the OP seems to have tried most of them).

I wonder if there is a way of modifying the acidity of a common inorganic acid by adding something else to the mix. Maybe use a solvent that's only part water, but that would involve adding organic compounds again."
posted by web-goddess at 7:09 PM on June 20, 2007

I have passed this on to my chemistry PhD dad. He's going to be away from the computer for a couple of weeks, but if this question is closed, and it's still useful to you, I'll email any answer he comes back with.
posted by i_am_joe's_spleen at 7:50 PM on June 20, 2007

It's the Biochemistry / Molecular Biology CRC. It definitely lists common buffers, but they're probably all carbon containing except for borate, which you've already ruled out.
posted by u2604ab at 7:50 PM on June 20, 2007

Ouch. This is a tough one. Is the issue with Carbon that you don't want reactive Carbon, or are you doing a spectroscopic assay that will pick up Carbon? If you can bear trying an organic buffer, I'd say go for a pyridine buffer if your issue isn't spectroscopic, but be careful, as aromatics sometimes intercalate into proteins and nucleic acids.
posted by yellowcandy at 8:54 PM on June 20, 2007

KClO4?

I've seen it used at pH ~ 3, so it might go higher. Can't find a pKa though. Or phosphorous acid? My googlefu is failing on the pKa info though.
posted by kjs4 at 10:49 PM on June 20, 2007

Google submits:

acid/base constants

Don't know anything about the reactivity of any of these though.
posted by kjs4 at 10:54 PM on June 20, 2007

I might sound completely stupid here, what with all the references to professional chemists, but is there some reason why Tris buffer can't be used? We* use it in immunohistochemistry all the time.

Immunology lab, personally, but most biological scientists do some form of IHC.
posted by kisch mokusch at 8:14 AM on June 21, 2007

Forget my comment, please. Didn't even notice the carbon bit. Jesus, I'm thick today!
posted by kisch mokusch at 8:16 AM on June 21, 2007

Response by poster: OP here. A few answers/clarifications/follow-ups:

yellowcandy: The reason that I can't use an organic buffer is because we're doing some ¹³C stable isotope analyses on the assay as well. Anything containing carbon would contribute an unacceptable amount of background to that analysis.

kjs4: Did you really mean a perchlorate buffer, or something else? The pKa of perchloric acid is -7, which means it's totally dissociated well before pH gets into the range we're working in. Phosphorous acid might be a possibility though -- it doesn't appear to be too nasty but it does slowly oxidize to phosphoric acid in the solid state, no idea what happens in solution. Also, I haven't had time yet to read through the table you linked to but maybe I'll find something there.

i_am_joe's_spleen: this question does seem to be open still, so if you happen to remember to ask your dad when he gets back I would appreciate it. (Based on my emails with some of you, there's been a lot of dad-asking on this issue!)

To the rest of you, thanks for your help. If/when I come up with a solution to this I'll post it back here in this thread, in case it will be useful to someone in the future, or if you're just curious about the solution to a random stranger's problem.
posted by harkin banks at 10:52 AM on June 21, 2007

Hi Harkin,

Yeah, I saw the pKa afterwards - it's just that it was used as a buffer in a paper I was reading when I saw your question. Not sure why they used it.

Possibly because it's mostly KClO4, and that effectively makes it a buffer ie. pH doesn't move much when acid or base added. Actually, that might be another option. Find a (very) weak acid, and just use that in it's pure form. I just read a disscussion here:

Chromatography Forum

about how pure WA or WB's can be considered buffers from a practical point of view, as they do buffer the pH. Actually, it would be worth asking that forum if they have any ideas. They work with buffers a lot for liquid chromatography, and might have some ideas.
posted by kjs4 at 5:23 PM on June 21, 2007