Hello,

I am wondering if anyone has ideas about separating acetic acid and TFA in a small molecule matrix by RP. I have tried an Alltech Prevail Organic acid column, 25mM KH2PO4 buffer at pH 2.5. The pka of acetic acid is 4.75 and for TFA it is 0.3. Using 100% buffer only as the mobile phase, acetic acid is separated but of course TFA is basically not retained. I have read about ion-pairing reagents such as tetrabutylammonium. Does anyone have experience with this reagent? Also, I plan to add a methanol gradient to elute the small molecule matrix after the acids have separated.

Regards,

Travis.

Before jumping to ion-pair chromatography (especially if you plan to add a gradient...be ready for long re-equilibration times), I would look at Phenomenex Synergi columns for your separation. The Synergi Hydro-RP and Polar-RP are specially designed for highly polar molecules (like organic acids) which are unretained on conventional C18 columns. I've used them for both glycolic acid (Hydro-RP) and lactic acid (Polar-RP) and have been quite happy with them. For my purposes, I was able to use 0.1% Phosphoric Acid as my mobile phase and got more than adequate retention, resolution, and peak shape.

If you have to use TBA as your ion-pair agent, use small quantities (2-10mM in most cases), try to find the purest grade reagent (ion-pair reagents are notorious for being rather impure, causing noisy baselines, and sometimes the impurities interfere with the peak of interest), and be prepared to dedicate those columns solely to this separation.

And, as usual, check out a copy of Snyder, Kirkland, and Glajch "Practical HPLC Method Development". They have a great section on ion-pair theory and method development, including determing how much to use when using X% methanol in the mobile phase.

Good luck. If you have any further questions, don't hesitate to ask.

Travis,

I think you're odds are low on achieving sufficient retention in the ion suppression mode. Provided you are determined to separate these anions on a reversed phase column (they're quite easy to retain and separate by anion exchange), I would simply use 2 mM tetrabutylammonium borate. You can't buy this particular salt but it's easily made by using boric acid to adjust the pH of tetrabutylammonium hydroxide (good quality reagent is available from Sachem and Fluka). It takes a fairly substantial amount of boric acid to drop the pH below 7 but the boric acid doesn't interfere with retention. You don't want to use tetrabutylammonium with a high affinity counter ion (such as chloride or phosphate) since this will interfere with retention of acetate. I would start with 10% acetonitrile but you may need to increase it a bit as trifluoroacetate is much better retained than acetate. If your sensitivity requirements aren't demanding, you should be able to get away with using UV detection but you can get significantly better detection limits via conductivity. The tetrabutylammonium borate eluent is suppressible, which provides very low backgrounds and high signal to noise ratios.

It has been a while and memory is dim, but I think we were able to do this on an ES Industries Aquasep with dilute phosphoric acid as eluant. If I remember correctly TFA was barely retained. Detection was at 205 nm. If nothing interfers you can get 1 ppm detection limits for acetic. You might get Matt at ES Ind. to loan you a column to try it. I certainly would not buy one based on my memory!

We did several acids too with dilute phosphoric on a Atlantis dC18. It gives good retention. In our assay, TFA was not included, therefore I can't tell how well it will be retained.

I'd recently run some analysis on a YMC Polyamin II and had a retention for TFA of appr. 5 min. Mobile phase contained small amounts of acetonitril (1%) and sulfuric acid. pH was around 2, which was no problem for the Polyamin II.
TFA doesn't look like a nice peak should do and I don't believe that acetic acid will be retained enough under these conditions. Maybe if sulfuric is replaced by phosphoric acid and pH is around 3 or 4 both acids will be retained enough for your purpose?

Regards, Andreas

Maybe GC would be a better choice.

ion chromatography works very well for these compounds. Dionex has an application with specifics on their website.