Chromatography Forum

In a reverse-phase HPLC system, what buffering system would be ideal to achieve a low-pH mobile phase (2.0) that won’t also depreciate the instrument like HCL would? ('ideal' = precipitation unlikely, well behaved).

In my experience, use of acetic or formic acid seems all around less problematic compared to a mobile phase composed of an inorganic buffering system where precipitation can occur if not careful. Unfortunately, the selection of organic buffers is rather slim compared to inorganic systems.

My question is this:

Where a pH lower than 3-4 is desired, can the pH of 0.1M acetic or formic acid mobile phase be adjusted with HCl, (such that HA/A- >10)? This affords a solution with poor buffer capacity, however, with the higher concentration (0.1M) of acetic acid, will fluctuations in pH even be measurable? Will the pH be expected to stay constant throughout the run?

Thanks,
PM

The pH of 0.1% TFA (trifluoroacetic acid) is below 2 (~1.95), and it's my favorite for general HPLC test. UV cutoff is 210 nm.

I'm concerned mostly about fluctuations in pH during the run. For many applicatations, a little change in pH is tolerable and largely remains a non-issue so long as well resolved, symmetrical peaks are eluting. My application hinges on knowing the pH at all times. I am rather new to the sport, and am finding out that pH monitoring of the mobile phase isn't such a trivial measurment, particularly when a pH gradient is used.

Lowing the pH with Trifluoroacetic acid is certainly more attractive than using acetic acid that is enhanced HCL where I'm not even certain a pH at or below 2.0 is even be attainable.

Cheers,

That precipitation fear is often overdone. If you stay away from dangerous (in this context) concentrations, precipitation is not only unlikely, it just doesn´t occur. Testing is very simple, also Tom just gave a link to an article on this: http://www.lcgcmag.com/lcgc/article/art ... p?id=97119

Also, as mentioned many times, at pH = 2 you have very good buffering toward acids and small amounts of bases (if you add considerable base you may reach a nonbuffered pH quickly) , regardless of what you use to attain pH = 2.

Now Cl- at that pH can be very incompatible with stainless steel.

As long as your hplc system is not designed to be used with HCl you should avoid this acid at low pH.
For pH 2 or lower you can use TFA but I prefer sulfuric or phosphoric acid. TFA isn't such a "nice acid" as one would assume - there is a thread about TFA (see TFA at 215 nm).

Whatever of these three acids you take for pH 2, calculate the conc of injected(!) basic compound against the conc of acid from mobile phase for injection volume. As long as sample matrix can be ignored and low volume is injected you should be on the safer side.

Use phosphoric acid. it is cheaper than TFA, no UV absorbance and it provides excellent buffering capacity at this pH.