Chromatography Forum

Hi all,

I have been recently developing HILIC methods, with UV detection (not MS). When browsing the literature, I very often come across the very odd "200mM pH 3 ammonium formate buffer". If I remember well from my studies, the pH of a solution of salt is independant of the concentration, and is the average of the pKa's of the two components. In the case of ammonium formate, the pH is 6.5.

So of course, to bring the pH down to 3.0 you need a HUGE amount of acid (I assume the pH is adjusted with formic acid, though it is generally no stated anywhere). In the end, you have a solution of formic acid, much more concentrated than 200mM, with a little ammonium, and I think no buffering capacity at all.

My questions are :
- is there any other interest than MS-compatibility to use ammonium formate in HILIC ?
- why call it a buffer when it is not a buffer ?
- to which chemical entity does the 200mM concentration refer to ?

In a word, how do you folks prepare a "200mM ammonium formate pH 3 buffer" ?

By the way, I used formic acid alone and it worked perfectly...

Dominique

Dominique,

1) You do not have to use ammonium formate if you do not use MS. Already 200 mM is too much for MS.

2) Actually you are wrong. I used the program pHoebus to calculate the concentrations needed to create 200 mM of the above solution. The program calculates up to 150 mM as it is mainly used for the creation of CE solutions. So it calculated that you need 148.69 mL of a 1M ammonia solution and 797.15 mL of 1 M formic acid (for 150 mM of ammonium formate at pH 3). The buffering capacity was 300 mM/L which is quite good and what expected as you can buffer in that area of pH with formic acid (read previous post about PH/buffering capacity and bibliography on the subject).

3) There was a huge discussion (see buffer preparation tread) about how you should prepare your buffer so I won't say what the usual practice is (that will generate further posts on the same subject). Read that thread for how you should prepare your buffer.

The reason that people use higher concentrations of buffered solutions with HILIC is because it decreases tailing that you might observe for some compounds with this mode of separation. If formic acid works just fine for your application (in terms of peak efficiency, reproducibility etc) then stick with it...

Kostas

The main reason for using such a high ionic strength is probably to suppress strong ionic interactions with ion-exchange sites on the column.

A volatile buffer is also proper for evaporative light scattering detection.

Seacrd:To help your understanding of the situation.... The formic acid will be neutralized with ammonia. The pH is clearly going to be acidic so long as the solution contains ammonium ion(which is not basic) and formic acid which is acidic. At this low pH there is but a vanishingly small amount of ammonia present. So a way to look at the situation is that the potential ammonia/ammonium buffer has no buffer capacity and hence will have no influence on the solution pH so long as all the formic has not been neutralized. Past the equivalence point the solution will be basic and the pH will be governed by ammonia/ammonium buffer and formate will be irrelevant. Therefore, in principle one can prepare an ammonium formate buffer just as one would prepare a sodium formate buffer and it will buffer well +/- 2 pH units from formic pKa, or +/- 2 units from Ammonium pKa.

As you state, In aqueous solution buffer pH is determined by ratio of acid to salt, neglecting effects of activity coefficient. I don't know what HILIC is, but if it involves a mixed aqueous/nonaqueous solvent this assumption may not be true. [/i]

seacrd,
It would be wise to refer to the entity doing the buffering when talking of molarity of a buffer (here formate).
To get a "picture" of what Bill said, take a book on chemical equilibrium and check the derivatization of the buffer index (capacity) formulars.
Now, formic acid alone at 200mM does not have a pH = 3, your statement that your chromatography worked with formic, alone, is equivocal.