Pressure problem with aqueous ammonium acetate eluent

[JR]

Hi all,
I have an LCMSMS method in negative mode that I used to run with water as eluent A and a 1/1 mix of acetonitrile and methanol as eluent B. This gave me some issues sometimes with the peak shapes of acidic compounds. I figured out it was a pH issue and added 1mM ammonium acetate to the water to fix the pH. I know ammonium acetate alone is not a buffer but that was sufficient to get rid of my problem without decreasing to much the sensitivity in the MSMS. The down side it that the pressure in the system increases very rapidly (from one sample to another) and the pre-column is quickly clogged up with some dark residue. Changing the pre-column allows to come back to normal initial pressure but as soon as I run a few samples, the pressure increases again. We thought about bacterial growth and we made a fresh stock solution of 1M ammonium acetate with 10% methanol and prepared a new eluent at 1mM ammonium acetate with 5% of eluent B in it. That did not help...so we are pretty sure the problem is linked with the use of ammonium acetate as we never had this problem with pure water before but we can't really find the root of it and therfore we can't find a solution. Has anyone experienced similar issue and solved it and how? Or does anyone has an idea that could help? Thanks a lot in advance.

[kerri]

You should filter the ammonium acetate solution. But, from what you're describing, it seems that the problem is coming from your samples. What kinds of samples are they? What kind of sample prep? Did you centrifuge them before injection?

Kerri

[usuchrom]

I need to know the brand of your LC

[JR]

The LC system is a Shimadzu UFLC.
The samples are secondary effluents from wastewater treatment plants. Samples are filtered through 0.45µm membranes before solid phase extraction, elution, drying and reconstitution in 1 mL solvent. We analysed the same kind of samples before, from the same place and there was no issue. The pressure problem occured exactly when we added ammonium acetate in the negative mode. The exact same samples give no trouble in positive mode (same pre-column and column used). We use water and acetonitrile as eluents in positive mode with 0.1% formic acid.
Thanks.

[usuchrom]

I propose a few things. First, make an injection of a blank using pure water (for 10 column volumes). And then, 10 column volumes with 100% ACN. If this fails, it could be damaged the rotor of the valve selector.

I usually work with the ammonium acetate phase. About: "and the pre-column is quickly clogged up with some dark residue", that dark residue used to be from algae growth in water (or water HPLC system itself.) Amber bottles used to slow the growth of these algae. Renew water bottles frequently.

[JR]

We just tried the same method but going up to only 95% of organic eluent instead of 100% as we use to do and that seemed to have solved the increasing pressure issue. So we think that the residue and the pressure increase were possibly due to ammonium acetate precipitating on the pre-column when going to 100% organic eluent. But this is surprising since the organic eluent does not contain ammonium acetate, it is added only in the aqueous eluent. Moreover, the organic eluent is methanol/acetonitrile (1/1) and ammonium acetate is supposed to be very soluble in methanol.
Anyway, thanks for you help.

[pcolin]

Hi all,

I'm currently facing similar problems. I've been using a 5 mM ammonium hydroxide buffer (adjusted to pH 9,2 using acetic acid) on two different systems. On one system i used to run a gradient ranging from 95% to 0% buffer (the other MP constituent being MeOH) on the other I performed isocratic runs with 65 % MeOH.

Both systems suffered from the mentioned pressure built-up. So far the only solution has been to change the inline filter-frit periodically.

Since high MP pH is crucial for my application and ammonium hydroxide buffer seems to be the only real buffering system availiable for LC/MS aplications, I'm forced to solve this issue.

Ideas are therefore more than welcome

[Uwe Neue]

What column are you using?

[pcolin]

Both applications were run on a Waters Acquity BEH C18 column.
(a dedicated column for each application)

I don't think the problem has anything to do with my column. As mentioned before, the sole solution has been to replace the inline-filter regularly.

Does anyone know whether there could be a incompatibility issue with PEEK or another frequently used material?

Kind regards,

[Bruce Hamilton]

I've used higher concentrations of ammonium acetate in PEEK systems for months without problems. I'm assuming that injections of clean blanks does not increase the pressure.

My suspicion would be that the ammonium acetate is solubilizing some component of your samples and carrying the ammonium or acetate salt into the guard. When you go to high organic mobile phase, the material drops out.

I would ensure the sample was in mobile phase, and centrifuged or filtered before injection. If you don't need to go to 100% organic, it would be preferable to stop the gradient at 80 or 90% organic.

[pcolin]

Pressure does increase on injection of clean blanks.
Even when the system is left in stand-by the system pressure increases.
I'd like to point out the fact that I've experienced the same trouble when I was running isocratic runs (65% MeOH - 35% NH4OH in water) on another system.
Fact is that something is being caught on my guard / inline filter. Since the system pressure built-up is not dependent on the injection of samples, I'm guessing an incompatibility between my MP and some LC component might be causing this.
Does anyone know of an incompatibility issue between high pH MP and frequently use LC materials?

[Kostas Petritis]

In the early days of UPLC, there were rotor coatings that would shed depending on the operations conditions but this might not be the case anymore.

Having said that, we do have our share of problems with ammonium salts (high pH) and pressure increases or pressure spikes when doing 2D chromatography. We did also found some evidence of crystals but it was unclear if those would had formed if we were using the system 24/7.

I also looked if there is some relation between crystalization and the use of ammonium salts, pressure and high pH and found some evidence that there might be a correlation (i.e. the article in the link below show that the solubility of ammonium salts decreases as the pressure increases). This might or not be relevant for the HPLC conditions, volatile salts and concentrations used but the fact that solvents are used as well might help with the crystalization process.

An interesting problem...

http://iopscience.iop.org/1742-6596/215 ... 012066.pdf

[Bruce Hamilton]

Does anyone know of an incompatibility issue between high pH MP and frequently use LC materials?

The most obvious would be any glass sinter frits in your solvent reservoirs. It would be very surprising for modern HPLCs to have materials that corrode with relatively-dilute ammonium acetate solutions.

Given that the pressure increases without injections, it should be possible to try "alternative source" materials for the mobile phase components and ascertain if they affect the rate of pressure increase.

I'd also make up some of the same composition as your gradient extremes, allow to stand for 24 hours, filter and determine if pressure increase rate is the same.