Chromatography Forum

Hi, I'm new in LC. When I prepared the organic mobile phase (acetonitrile, 0.1% formic acid and 2mM of amonium acetate) sometimes a white precipitae appears at the bottom of my botle. I'd tried to make a fresh amonium acetate buffer but still there are some white spots. Help me please.

If there is no water in there, I suspect that the solubility of ammonium formate and/or ammonium acetetate in pure ACN may be low (but I don't know of any published values).

What grade ammonium acetate are you using? What are the impurities in the acetate (lot analysis or cert of analysis should tell you)? It could be the acetate/formate precip'ing out, but it also doesn't take much of an insoluble impurity to make that "sinking haze". I would ultimately suggest filtering your mobile phase after preparing the buffer. It shouldn't affect your final molarity much.

Whereas I have been saying on occassion that chromatography is an art, I really suggest that one keeps the art to a minimum. In this case it would mean that it is better to find out why something is precipitating (does this white stuff readily dissolve in H2O?) and then preventing it. Maybe just adding some water will do the trick? (You probably add an aqu part later anyway?).

Like Tom I do not know of any published values but ammonium acetate is sparingly soluble in ACN. I believe the late Dr. Neue said to dissolve the salt completely in 5% water, then bring to volume in ACN, but I can't find the link anymore.

Yes, but if the precip is an impurity... why dissolve it? I like to know the answer... not just make the problem disappear. If it's due to the acetate/formate not being soluble then adding water is a fine solution. But if it's due to an impurity in the acetate I would not want to just dissolve it. If I couldn't answer the question of insoluble salt vs insoluble impurity.... I personally would filter the solution before use.

What if you reduce the concentration? Does the insoluble haze still form? There is definitely a fairly simple way to get an answer to this problem.

Filtering without knowing what and what fraction you are taking out is not science.

if it's not an impurity, you would be changing your percentage ammonium formate if you filter it out.

From http://www.emmert-analytik.de/HPLC2006_SanFrancisco.pdf:

...However, the limited solubility of ammonium acetate in commonly employed organic solvents makes it impossible to prepare solutions of more than ~0.05% in pure acetonitrile...

From http://www.lcresources.com/discus/messa ... 20030741pm:

...I have never been able to get even 10mM ammonium acetate to dissolve into such high acetonitrile concentrations (85-95%). Even if I dissolve it in the aqueous portion first, then add the ACN, I get phase separation that appears to be two immiscible liquids.

I should learn to follow my own advice and always search both the current Forum *and* the archives!

For those of you posting about "not knowing what your filtering isn't science" and changing the molarity by filtering out solute precip. I plainly said to find out. But to add water changes the mobile phase. It'll still work possibly, but so wouldn't slightly less concentration, possibly.

Your going to have to change something. I'm sure someone will argue adding water is less of a change than reducing the acetate, but you would be guessing unless you tried both ways. My point was if you needACN w/ ammonium acetate and you have a precip., I would filter... because adding water is no longer ACN / ammonium acetate. Please don't make me argue the finer points of my suggestion. That's silly.

I haven't done any HPLC for quite a while (thank the lord !) so forgive me for what might be a simple minded question - how does filtering out the precipate / haze / undissolved solid provide any information about what it is ?

Under this particular set of circumstances, and given the information that the HPLC experts have dug up I would think that adding a few percent of water would be a pretty definitive test for undisolved ammonium acetate.

Peter

Filtering the haze doesn't do ANYTHING but give u filtered buffer that won't harm your precious LC. To me that's top priority. The rest is up to you, there are numerous solutions.

If you really r that bent your concentration. Titrate. Or if u can add water and know it's not an impurity then add water if your method allows. I'd still filter after the precip. forms no matter what you decide.

Haze and precipitates are nothing? Great science! (This is the last statement from me on this).