Use of Glacial Acetic Acid in Mobile Phase

[adam]

Hey All

I have come across a few methods recently that use Glacial Acetic Acid as the mobile phase additive. So I am curious to poll the forum: Do you see any situation where glacial acetic acid has a particular benefit over other acids.

Although acetic acid is one of the volatile acids - so it has that benefit - I tend to almost never use it, simply because it is a weaker acid, and so you need to add more of it to get the pH down. Personally I tend to go with formic acid most of the time. But since I'm seeing a number of methods that use acetic acid lately, I am starting to second guess myself and I'm wondering if there may be something I'm overlooking. So my bottom line question: what situations, if any, would you consider acetic acid to be the best option?

(and by the way, I am talking about reversed phase here)

Any thoughts are welcome.

Thank You

[Consumer Products Guy]

We use acetic acid as pH modifier/control in our aqueous mobile phase quite a lot. This is going on 35 years now....typically that acidic aqueous mobile phase is used with ACN or methanol.

[Andy Alpert]

Look up B.S. Welinder and H.H. Sorensen, J. Chromatogr. A, 537 (1991) 181. This paper discusses the use of acetic acid as just another organic solvent for eluting proteins from reversed-phase columns.

[adam]

Thanks for the reference Andy. Unfortunately, I don't have access to get the paper. Is it possible you can summarize their observations in a few bullet points - with respect to what Acetic Acid does that is unique in this case.

Thank You
Mark

[Andy Alpert]

Sample: Interleukin-1-beta, a "sticky" protein.
Column: Dynosphere, a PS-DVD material. This performed somewhat better than a C-4 column with a regular gradient of ACN with TFA present [NOTE: TSK-Phenyl performed better for membrane proteins from erythrocyte ghosts].

Various combinations were tried: Increasing acetic acid in ACN, in propanol, and in water. The best combination depended on the sample. After the interleukin, they proceeded to analyze pancreatic extracts and extracts of E. coli engineered to produce human growth hormone. The authors speculate about acetic acid's role in disrupting hydrogen bonds, but formic acid generally didn't work as well as a chromatography additive, so the reason for acetic acid's good performance remains obscure.

[adam]

Andy: Is it possible that there was just something unique about that particular application. That reference dates back 23 years ago. And if this hasn't been observed or discussed since then (or at least not frequently) is it possible that this was a "fluke" of some kind. I hate to sound like I'm handwaving. But let me ask this way: You deal with proteins all the time; have you found there are times where acetic acid works better than other additives.....often.....rarely....almost never?

I'm also wondering - and this is for everyone - if anyone has found any unique benefit of acetic acid with respect to small molecule separations.

Much Thanks
Adam

[Andy Alpert]

I agree that Benny Welinder's paper was a one-off. Perhaps the reason has nothing to do with the chromatography. A couple of years after Welinder's paper came out, we were auditioning HPLC gradient systems from various manufacturers, trying to decide which one to buy. The manufacturers assume that most customers will be using C-18 columns with a gradient of ACN in water. Accordingly, we challenged the systems with a gradient from 0-50% acetic acid in water. Half of the systems couldn't handle this, on account of the considerable viscosity.

One application involving proteins that does benefit from acetic acid is a method that I introduced at ISPPP 1996 and which since 2009 has been adapted to separation of histones. This involves applying proteins or peptides to a weak cation-exchange column at a pH where it has (-) charge (say, 10 mM ammonium acetate, pH 5.0) and then running a gradient to 10% acetic acid or 0.5% formic acid. This decreasing pH gradient uncharges the WCX material and the proteins elute in a volatile, MS-compatible solvent. The histone labs put 60% ACN in the starting mobile phase as well in order to superimpose a significant degree of hydrophilic interaction. Do a search on Google Scholar with the terms "WCX-HILIC" AND "histones", between 2009 and 2014.

Acetic acid and small molecule separations: Out of my realm of expertise.

[danko]

Adam wrote

because it is a weaker acid, and so you need to add more of it to get the pH down

Adam, if you need lower pH than 3.5 then don’t use acetic acid (acetate buffers). Tt’s as simple as that. One of the arguments for using acetic acid is precisely because it’s a weak one and it’s buffering at relatively high pH values.
Another important factor is, its relatively chaotropic nature which is quite useful indeed – especially in protein separations and protein chemistry in general.
Hope the above answers some of your questions.

Best Regards

[mattmullaney]

Well, not to be a "nudge," but being one probably, anyway...it's already been noted that acetic acid is Volatile. An advantage in some applications.

Next...what better to pair with acetate to make buffer solutions from pH 3.6 - 5.6 or so?

Based on a cursory glance at ScienceDirect (Elseveir), glacial acetic acid (like ethanol as opposed to methanol) seems to work successfully quite a bit in chiral separations.

Oh heck, I don't know. Could be because phenomenologically-speaking, it happens to work. If the selectivity is there for both formic acid and glacial acetic acid...I guess you could pick and choose your preference.

There's a decided advantage for acetic acid in TLC, but that wasn't the question asked.

Otherwise, other acids have a decided advantage over acetic acid in terms of UV cutoff...but that is only "one case."

I'm done now. Glacial Acetic Acid is less expensive than other acids, certainly...I'd sort of not like that to be a "real" part of the answer. But then, it's not an unimportant consideration.

This was an interesting chapter, too:

http://www.sciencedirect.com/science/ar ... 0133000070

but it didn't help much this time.

Please see what you think, and thank you.

[lmh]

I'd caution about using high percentages of acetic acid in LC-MS. We had a method using 10% acetic acid in an Agilent single quad MSD. It worked beautifully. I think the analyte was being measured in negative mode. After a week or two we tried some positive mode work and realised the instrument had lost nearly all its sensitivity.

Agilent's explanation was that the inside of the glass ion transfer capillary, which links the spray chamber with the vacuum part of the instrument, had probably got coated in acetate, turning it into a sort of gas-phase ion-exchange column that was mopping up any positive ion entering the instrument.

We did manage to get the sensitivity back by copious washing of the ion-transfer capillary, but given that it's stupidly expensive, made of glass, has delicately platinum-plated ends, involves venting the instrument to get it out, and getting it in carries significant risk of messing up the little canted coil springs that hold it at each end, meaning yet more expense and fiddling, it's not something I'd want to do regularly.

We now stick to 1% maximum of acetic acid in general, though I usually use formic acid as my default.

This may be a uniquely Agilent issue, other manufacturers using metal ion transfer tubes.