Chromatography Forum

Dear all;
5% will be too high for the concentration of Ammonium Acetate in the mobile phase? I find a hplc method using this percentage in a research paper.Before my study, I want to check whether the concentration is suitable.
Best regards!
Terry Z

In theory it is possible to have 5% buffer in water as solubility of ammonium acetate is around 150 g/100 ml of water. You might have problem with layer separation or crushing buffer if you use too much ACN.
Make sure that it is 5% of the ammonium acetate and not 5% of 100 mmol buffer or something like that.

I dissolve 50g ammonium acetate in 1L water.
As you said, when I mix ACN and buffer(3:1), the mixture divide into two layers, but the rate of layers is not the same as ACN :buffer(the layer underside is larger than upper).So I changed the proportion to 1:1, and got a pellucid mixture.However,when the room temperature changed from 25 to 8 centigrade, the mixture(1:1) separate into two layer again.
Can you explain this phenomenon for me? Thank you very much.
In addition, my hplc column's lifetime is very short(about 100 injections) using this mobile phase. After 100 injections, the peak become broad, then backpressure increase drastically.Did high concentration salt buffer cause that?

This is related to solubility of your buffer solution in ACN, at lower temperatures it is lower. Also you might not see the layer separation.
What is your column, detection technique and compounds? May be you don't need such high concentration of buffer.
You might be always exposed to this potential problem at such high buffer concentrations.

Don't forget that your solution of ammonium acetate, on its own, is not a buffer. You need to add acetic acid to it to make it a buffer.

I'll refer you to the HPLC troubleshooting guide by Uwe Neue. Search for "HPLC troubleshooting guide" and pdf in google.

For most HPLC assays, I would recommend using 100 mM or lower.

For UPLC/MS/MS assays, only use 5 mM or lower. Over 5 mM, you start to run into suppression issues and your source gets dirty very quickly.

JayVee, if one dissolves a high grade of NH4OAc in water the pH will be close to 7. Appreciable buffering is expected around 4.76 and 9.26 (the relevant pKa from my lit.). you can add any suitable base or acid to get into these buffering ranges, HOAc is not the only species one needs to add.

JayVee, if one dissolves a high grade of NH4OAc in water the pH will be close to 7. Appreciable buffering is expected around 4.76 and 9.26 (the relevant pKa from my lit.). you can add any suitable base or acid to get into these buffering ranges, HOAc is not the only species one needs to add.

Thanks for the clarification. HOAc is convenient to make the buffer at pH 4.76, since one would just need to add the same amount of HOAc as there is of NH4OAc (equimolar solution).

I don't know how that would affect the solubility of NH4OAc, however.

What type of method is this using 5% Ammonium Acetate? There may be other alternatives.
For reverse phase, I've understood that 50 mM is enough to buffer effectively.

Maybe, I need to describe my method more distinctly:Mobile phase was A ( buffer of ammonium acetate(5%) and copper acetate(0.02%),adjust to pH 5.0 with alacial acetic acid) : B ( methanol ) =83: 17; column temperature was set at 25℃; the UV detector was set at 270nm; the flow rate of mobile phase was 1.0ml/min and inject volumn was 10ul. It is a derivatization method detecting Ethambutol hydrochloride.To elute more hydrophobic impurity in the tablet, we add a gradient of buffer:ACN(50:50) after elution of Ethambutol hydrochloride. HPLC Column we used is Supelco Ascentis 3u C18 150*4.6mm.
Best reagards!
Terry Z

If you get layers (what is pellucid) you may have been victim of a printers mistake, maybe they meant 0,5%?

JayVee, it is correct that if you add equal moles of HOAc and NaOAc your pH should be ~ at the pKa, but NH4OAc/HOAc? Maybe some math wizz out there will do a calc to show what is needed?
zlb215, do you know how much HOAc you added to get a pH of 5 (from pH = 7 NH4OAc?).

How different is ammonium acetate vs. sodium acetate?
The ammonium is a little more acidic so you should add a little less of the acetic acid and then adjust the pH.

No difference. The pKa's of the ammonium ion and the acetate ion are miles apart. the optimum pH for an acetate buffer is pH 4.75, independent if it is made from ammonium acetate or sodium acetate. And you will always prepare from equal ,olar amounts of ammonium acetate and acetic acid, as pointed out by JayVee above.

yeah i figured it wouldn't matter

This nicely shows my point, it is not trivial for people who do not do these calcs every day, me included. I am not masochistic enough to sit down and do the calc. Now most of us agree that calculating the amounts and weighing is more accurate than using a pH meter. The calculator,

http://www.liv.ac.uk/buffers/buffercalc.html

doesn´t have ammonia so no help here. But: One can do a little mental work. The NH3/NH4+ present at pH 7 will "soak" up H+ if acid is added to the neutral NH4OH, Na+ can not do this. So what Uwe says is opviously correct, the optimum range is independent (note that I already indicated that above), but the amount (moles needed) to prepare this will theoretically not be the same for the two cases.