Chromatography Forum

I recently implemented a method transfer for determining residual TFA/Acetate on a C18 column, and it works brilliantly. I'm just kind of curious as to how it works, though. Are TFA/Acetate actually retained by hydrophobic interactions, or is there something else going on?

The conditions are:

0.1M pH ~2 phosphate buffer with 2% MeOH
1.5mL/min flow rate
C18 column, 4.6x250mm, 90-120A pore (haven't tried anything larger, honestly), 5µm packing.
UV Detection at 210nm

k' values for both compounds are less than or equal to 1, but reproducability and linearity is easy to achieve. Resolution is easily better than 2 with a few of the columns I've used, but it seems better on vanilla C18 columns (like a YMC ODS-A) than on, say, an aqueous C18 column (like a Phenomenex Gemini). The TFA peak is pretty asymmetrical, kind of right-triangle looking, but the Acetate peak is perfection on a good column.

I could see the acetic acid being retained by reversed phase mechanisms, but the TFA is both highly fluorous AND deprotonated so it's gotta be pretty polar too. Anyone have any ideas about how the TFA is being retained?

There are a lot of possibilities:

1. TFA *is* somewhat hydrophobic. In fact it functions as a weak ion-pairing reagent.

2. Many C18 columns, espcially the "vanilla" ones like the ODS-A have a lot of residual silanols (i.e., only partial coverage of the silica surface), so you may have some hydrogen bonding going on.

3. k'< 1 isn't a heck of a lot of retention; that and the poor peak shape for the TFA would make me *very* leery about problems with that method in the future. For what it's worth, the FDA's "Guidance for Reviewers" of validated chromatographic methods suggests that k' values should be >2.

You can try analyzing TFA using this method with Unison UK-C18:
http://www.imtakt.com/TecInfo/TI248E.pdf

Almost forgot to note this:

viewtopic.php?t=9851&postdays=0&postord ... d&start=15


pepidemetdev, you could solve the problem raised in the link by looking at the retention of these acids under different pH, lets say up to pH 5 or 6. You could also answer at least part of your question that way.