Buffer solutions based on cation or anion?

[kmichel]

I'm following a HILIC method and trying to make a 50 mM ammonium formate buffer at pH = 3.0. According to the method I'm following, anything less than 50 mM formate buffer yields poor separation. I don't have ammonium formate, but I do have formic acid and ammonium hydroxide and I'm using these to make the buffer.

I added 2.3 g formic acid (MW = 46) to 1 L to get 50 mM formate, then added ammonium hydroxide at a concentration of 7.5 mM to get to pH = 3 (verified using a calibrated pH probe). However, my separation was poor. I always thought that if I were making a formate buffer, the concentration of the buffer was based on the concentration of the formate. However, I just read on the "Buffer Preparation FAQs" at SIELC that "the buffer molarity is the number of moles of a cation portion of the buffer". This would imply that my ammonium formate buffer is not 50 mM, but actually 7.5 mM. Which is correct?

[tom jupille]

In general, the concentration that matters is thst of the species doing the buffering. At pH 3 that would be formic acid / formate.

That said, why not try doubling the concentration and see if the peak shape improves?

[HPLC chemist]

I agree with Tom. Try 100 mM. At pH 3 all the side groups should be protonated so you will only have 1 ionic form.

[rb6banjo]

The pH is always dictated by the strongest acid in solution and buffers are in theory, independent of dilution. If doubling the concentration of the buffer in your mobile phase has an effect, then it's got something to do with the way it changes the interaction of the analyte with the stationary/mobile phases. Doubling the concentrations of your buffer constituents won't change the pH or the degree to which the conjugate acid is protonated or not.

A 50 mM solution of formic acid (pKa = 3.77) should give a pH that's somewhere close to 2.5. Adding the hydroxide base brings it to 3. Doubling all of the concentrations should still give a solution of pH = 3 and the relative amounts of formic acid, formate, ammonium, ammonia, and the concentration of protons will still be the same. All you've done is change the ionic strength of the mobile phase.

[tom jupille]

All you've done is change the ionic strength of the mobile phase.

Which is exactly what I suggested. Ionic strength effects are a significant factor in HILIC.

[HPLC chemist]

Again I agree with Tom (twice in 1 day ). Remember, there are other molecules present in your extract that can consume the ionic buffer. In this case you want to swamp the molecule of interest so you only have 1 ionic form.

[HPLC chemist]

If this doesn't work you're allowed to issue an expletive under your breath .