Chromatography Forum

Hi folks,

Just to be sure, wanted to double check the following with you;

Formic acid has a pKa of 3.77 and Ammonia 9.25.

(1)
Two buffer ranges seem to apply when an ammonium formate buffer is created, namely 2.7-4.7 and 8.25-10.25. The first range is to set ammonium formate with formic acid untill the correct pH is reached, the latter is set by adding ammonium hydroxide to the ammonium formate untill desired pH is reached. Correct?

(2)
Ammonium formate is actually a salt, not a buffer. In order to create a buffer with this salt, one needs to add eather formic acid, or ammonium hydroxide, thus would it not be better to call such a buffer either a formate buffer, or an ammonium buffer?

Thank you for thinking with me on this.

To (1): Perfectly right. You do not have to start from ammonium formate. You may use fromic acid and ammonium hydroxide to get buffers at both pH ranges.
To (2): Correct, too. I'm actually used to speaking just of "formate", "ammonium", "phosphate", "acetate" etc. buffers. Usually the counterion is not very important for the chromatography. And remember, you may also create a formate buffer from sodium formate. Or an ammonium buffer from ammonium phosphate...

Well the counter-ion does influence HILIC a lot, where the counter ion reduces secundary (ion-exchange-like) interactions with the stationary phase. For more details see: http://www.chromatographyonline.com/lcgc/data/articlestandard/lcgc/112008/501656/article.pdf

That's why it's important for me to do this correct.

HPLCaddict,

This is a basic question and I feel embarrassed to ask, but are buffers (such as ammonium formate taken to the buffering range with ammonium hydroxide) only buffering the pH? With the exception of ion-pairing, there is nothing going on with the buffers but to adjust the pH?

Ron J.

With the exception of ion-pairing, there is nothing going on with the buffers but to adjust the pH?

To a good first approximation, true. In practice, "secondary" effects can sometimes have an impact (for example, ammonium can arguably act as a competing base to tie up active silanols).

The ions from the mobile phase electrolytes also ion pair with charged groups in the stationary phase in addition to the silanols. This can influence retention and selectivity dramatically. See my paper in Anal. Chem. 80 (2008) 62 for examples. It takes about 20 mM salt overall to provide enough counterions to make a complete electrical double layer on the surface of the stationary phase.

Would this only be the case for certain stationary phases? In the case of a RP C-8 or C-18 column (i.e. Zorbax Eclipse C-8), there really wouldn't be anything else available for interaction, right?

I will have to see if I can find that journal...thanks.

Ron J

Regarding silanolate interactions with ions there was not much difference between Waters´Atlantic silica and Zic-HILIC (in experiments I did with radioactive ions). When we discussed this here before a conclusion seemed to be reached that all silica based columns can display "silanol" intractions.

rwjohnson wrote:
HPLCaddict,

This is a basic question and I feel embarrassed to ask, but are buffers (such as ammonium formate taken to the buffering range with ammonium hydroxide) only buffering the pH? With the exception of ion-pairing, there is nothing going on with the buffers but to adjust the pH?

Ron J.

Although late I think my answer will help others. That is exactly what it is. A good buffer is not supposed to interact with any of the components of the reaction mixture other than maintaining the hydrogen ion concentration. The interaction should be limited to weak acid and its conjugate base and vice versa. If you think the buffer is interfering with the chromatographic separation or an assay, then you have to look for another buffer that does not interfere.