Residual Solvents Acetic Acid by HILIC?

[sdegrace]

We have a project where we have to quantify residual acetic acid in the final product. We are required to exactly quantify it, unfortunately, that isn't negotiable. We expect it to be well below the ICH limit.

GC has proven to be very troublesome and in fact HSS GC is completely unsuitable although it works well for acetone, the other solvent we need to quantify. Due to the nature of the material (it contains a proteinaceous matrix) direct injection poses problems to say the least. The matrix and the product are water-soluble and we have been preparing the samples dissolved in water, although we have tried DMF and DMSO. In any case, I am given to understand that problems with HOAc in GC residual solvents methods are inherent and most labs don't try and do acetic acid this way, LC methods being more common.

I know I can do a reverse phase method that will retain acetic acid and I'm keeping the option in reserve (in fact, I have a promising-looking method in-hand), but I'm really intrigued to try HILIC instead. I'm hoping that the various "garbage" from the product and the matrix might be non-retained giving me a nice clear look at the HOAc (and maybe the acetone?) without gumming up the column. Just off the top of my head I was thinking of trying a 50 mM phosphate buffer pH 2.5 for the aqueous mobile and acetonitrile for the organic mobile and run it at maybe 80 or 90% organic (try mixing the mobiles on those proportions first to make sure nothing precipitates).

I have no experience with HILIC for this or any other application but I'm keen to try - does anyone have any comments or advice, especially regarding whether this application is really suitable?

Thanks,

Stephen

[tom jupille]

Another possibility is ion-exclusion, using a hydrogen-form cation exchange column from Hamilton, Dionex, or BioRad (to name three that come immediately to mind). The mobile phase is dilute sulfuric acid. Formic acid elutes first, followed by acetic, propionic, etc. Acetone, alchohols, etc. are more strongly retained. Proteins would probably bind tightly (so either a cleanup or guard cartridge would be necessary).

[SIELC_Tech]

see separation ofo acetic and formic acids on Primesep SB column with UV detection:
http://www.sielc.com/pdf/SIELC_November_2006.pdf

[Tobias Jonsson]

Stephen,

HILIC separation is indeed suitable for your problem and probably the most sensitive if you have an MS. The protein matrix can be a problem though and need to be precipitated before injection (we often use cold, acidified acetonitrile).

Phosphate buffer is not soluble in 80-90% acetonitrile. Even 70% can be too much. Use a buffer with high solubility in acetonitrile.

SeQuant has a couple of application notes (No. -12A and -13A) for different small organic acids, none of them for acetic acid though. But I think you are experienced enough to get the picture.
You find them at:
http://www.sequant.com/applications

You can also mail your HILIC questions to SeQuant at info@sequant.com

[sdegrace]

Thank you everyone for your advice and suggestions!

Tobias, what would you suggest for a buffer? I had heard suggested ammonium acetate or ammonium formate, although if I'm looking for acetic acid I have a visceral dislike of delibrately adding acetate.

I harboured the hope that I could get away without worrying about the protein matrix but I think you're right that we will have to precipitate it. I am also toying with the idea of doing centrifugal filtration (actually, this might make the GC method slightly more viable too) if I can get a suitable filter.

Unfortunately detection will have to be by UV but I think it's still worth a shot.

Stephen

[SIELC_Tech]

you can try HILIC ACN/water/sodium phospate pH 6.5 (watch for solubility of buffer). For HILIC you need to keep you pH above 4 in order to have acetic acid ionized and more polar. You can also try to do it without buffer, but my guess that proteins will interfere with your analysis.
With Primesep SB column and sulfuric acid in the mobile phase you can repel proteins which should come before void. Here is example of what happens to proteins on a similar column (mixed mode anion-exchange)
http://www.sielc.com/Technology_DirectP ... lysis.html
this approach is valid for Primesep D and Primesep SB. You can use phosphoric or sulfuric acid for acetic acid analysis on Primesep SB column. You will need very pure solvents and acid in order to see and quantitate acetic acid with 205-210 nm UV.
.

[Tobias Jonsson]

I understand your concern about acetate buffer. This is usually our first recommendation, but as alternative I would suggest ammonium formate in this case.
You can use pH to tune the dissociation and by that retention of acetic acid, but the full pH-range should be applicable. If you will use the precipitation step I would suggest formic acid for the acidification.

[SIELC_Tech]

Tobias,

And what you would suggest as detection technique for HILIC with ammonium formate to determine acetic acid? Unless you know something I don't, this is impossible with ammonium formate or formic acid, no matter what HILIC column Sequant's, SIELC's or Dionex'
The only choice to see it is no buffer at all or UV transparent buffer like sodium phospahte pH 6.5.

[XL]

As mentioned above by others, there are several ways to retain and analyze acetic acid on IC columns, RP columns, HILIC columns, and Mixed-Mode columns. I would try HILIC column for acetic acid only if I have to do so.
Attached is the information on Acclaim Mixed-Mode WAX-1 column (Dionex) which demonstrated the separation of a series of organic acids (see Figure 7): http://www1.dionex.com/en-us/webdocs/48 ... 021407.pdf

The mobile phase is phosphate buffer.
The K' for acetic acid is ~4.

Hope it will help.

[HW Mueller]

XL, your link doesn´t work here. It could also be instructive if you told us why you would do HILIC only if you had "to do so".

[XL]

HW,

To analyze acetic acid in HILIC mode, we need UV transparent mobile phase. It is advisable to use some kind of pH control for ionic analyte to ensure reproducible result. Thus phosphate buffer with high organic solvent content is an obvious choice. we also need acetic acid in its anionic form to maintain high polarity so that it can be efficiently retained on the HILIC column. As SIELC_TECH pointed out, pH6.5 phosphate buffer is a good choice.

Now, we need to worry about potential precipitation problem during the run. There are many kind of HILIC columns in the market. Some are more polar than the others. Thus the organic solvent content requirement in the mobile phase to ensure the adequate retention of acetic acid depends on specific HILIC column chemistry (from different vendors). Therefore, the precipitation risk varies. In addition, I prefer to use "greener" method.

I hope my confession of my earlier statement makes some sense.

Here is my strategy for analyzing highly polar analytes using HPLC method:

For highly polar anionics: Mixed-Mode Anion-Exchange column (Dionex, SIELC)
For highly polar cationics: Mixed-Mode Cation-Exchange column (Dionex, SIELC)
For highly polar neutrals: A HILIC column (Many vendors)

Sometimes, RP columns work, too. And a wide range of IC columns are best suited for small anions and small cations.

Regarding the link, I could open it from both my home and work computers. you might need to copy it to the web address to open it.

Thanks for reading

[HW Mueller]

Ok, so that´s a personal aversion, like my strong dislike of TFA.