Removing precipitant in column

[marine_micro]

Hi! I'm new to the forum and to LC in general...so greatings to all!

I have a problem regarding precipitation of ammonium acetate in my column. I am running 5mM ammonium acetate buffered water and acetonitrile using a Zorbax Eclipse C-18 2.1x50mm, 1.8 micron, 1200bar column. I am running several hundred samples and have found that the pressure goes up by about 100bar per 24hour period. I'm now committed to using the buffered water as over half of my samples have been run but the column won't last the distance if I can't figure out a way to remove this precipitation.

I have tried running MilliQ water with no ammonium acetate over 48hours. This reduces the pressure about 80bar in 12hours and then nothing more for the remaining time.

Does anyone have any suggests of how to remove this precipitant from the column?

[Consumer Products Guy]

I'd use 80% water, 20% ACN. Pumping 100% water changes the wettability of the phase, sometimes called phase collapse.

Did you pre-mix your mobile phase and then filter through 0.45u membrane?

[tadah]

You might want to include a short wash step (100% A) followed by a short column equilibration step at the end of each run.

[marine_micro]

Thanks for the comment on phase collapse (good to know!), and yes... mixed and filter through 0.2um membrane.

The HPLC I'm using has a column oven and the column can operate at temperatures up to 60oC (<40oC is optimal for long term survival of the column). I'm wondering if increasing the column temperature and running 80:20 water and ACN might work to shift the ammonium acetate. Any obvious problems in try this?

The other idea I had was to run 100% methanol to wash the column, since ammonium acetate is not soluble in 100% ACN but will go into methanol.

Thanks for any suggestions or comment. I really appreciate it!

[HW Mueller]

What makes you think that ammonium acetate precipitation is causing that, not crud in your samples?

[lmh]

to differentiate between insoluble-for-ever-crud and precipitation in column, you can put a small filter just upstream of it to catch gunk. This is good for your column anyway.

[marine_micro]

Hi, at first I though it might be my samples. I work with unknow secondary metabolites from bacteria so it is completely possible. The samples are filtered through a 0.2um regenerate cellulose membrane. The samples are kept on an automatic sampling system that is chiller 10oC for up to 24hours. There is the possibility that some of the later sample processed precipitate during this time.

What makes me think it is the ammonium acetate is the consistency with which the pressure increase, even if I am running water blanks. Secondly, I think this is the dead give away, I did't filter my buffered water daily, but when I do put a new batch of filtered mobile phase in the pressure does not increase over the next 24hour of run time. Once I notice this I started filtereing my mobile phase daily and the pressure increases stopped. Now I'm just trying to reduce the amount of precipitant in the column.

Also, I did have an in-line filter installed upstream of the column (before I figured out about filtering the mobile phase everyday). It did its job and pressure bulit up in the filter not the column.... but when I changed the filter the high pressure remained. The in-line filter unit has developed a clog that was not associated with the changeable filter membrane.

[Consumer Products Guy]

Interesting, I just used an acetate buffer for the first time Thursday and overnight myself, following a Dionex application note. The mobile phase was 0.10 M ammonium acetate in water (yes, 0.10 M) 70% by volume, and acetonitrile 30% by volume. I ran samples overnight, then washed the column today with 70% water/30% acetonitrile. I didn't notice any pressure buildup.

[marine_micro]

I used to run the same method/samples/mobile phase etc. on a different HPLC and column for weeks at a time... and never had any problems with the pressure. It has only started to occur once I moved machines and changed columns. Interestingly though... I've been filtering my buffered water twice daily and the pressure is slowly starting to decline (30-50bar per day decrease).

Might be hope yet to get through these samples!

[tadah]

It sounds more and more like an instrument or column problem. I think you need to check the tubing, injector, UV flow cell, column and other parts systematically for significant background pressures. I had problems with a column/mobile phase heater before and it created a lot of obstruction to the flow.

What happens if you just run injections of plain water over the same period of time? Do you see the increase in pressure?

[marine_micro]

Shortly after this started the the local technical rep suggested checking the factors associated with the instrument and when checked the background pressure was negliable.

Yep, tried injecting water only samples over 24hours and had the same increase in pressure over time.

I did just go and check my old column and it was 3 micron compared with my current 1.8micro column. This could have impacted the speed at which the precipitant acculumated and had an effect.

I think the reason might come back to one of the first question I was asked in this thread... Did I filter my mobile phase? Although I did, if something went wrong and unfiltered mobile phase got through, this could explain why it happened.

[HW Mueller]

I don´t understand all your explanations, but it seems to me that what you are seeing is not a precipitation of NH4Ac, rather it is some crud in the mobile phase or part of the system. I don´t recall either that NH4Ac would recipitate under the given conditions. It is easy enough to check that anyway (except if you get super saturation).

[danding_cr]

Hi, my thought about the precipitates is probably caused by ammonium acetate. I realized 10mM ammonium acetate would crash out from acetonitrile solution. So I wonder if this is the case: when you run the mobile phase containing 5mM ammonium acetate in water and gradually increasing acetonitrile concentration, ammonium acetate might precipitate when ACN concentration reached to a point (e.g >90%).

[HW Mueller]

We are discussing the horses mouth without looking at it again. Take a look at:
http://chromatographyonline.findanalyti ... rticle.pdf

With such a low concentration of ammonium acetate one will have difficulty to precipitate it from a reasonable mobile phase.

danding_cr, just because you couldn´t dissolve ammonium acetate in 100% acetonitrile (according to your statement in another chain) doesn´t mean that all water/acetonitrile/ammonium acetate mobile phases will cause trouble.

[danding_cr]

We are discussing the horses mouth without looking at it again. Take a look at:
http://chromatographyonline.findanalyti ... rticle.pdf

With such a low concentration of ammonium acetate one will have difficulty to precipitate it from a reasonable mobile phase.

danding_cr, just because you couldn´t dissolve ammonium acetate in 100% acetonitrile (according to your statement in another chain) doesn´t mean that all water/acetonitrile/ammonium acetate mobile phases will cause trouble.

Thanks for pointing this out as well as the article.