Chromatography Forum

Anybody eve use ammonium acetate beffer as one of the mobile phase solvent? I use it in one of my method, it is pH 6.7 ammonim acetate, 0.02M. I found this beffer or solution precipitate from time to time, after the solution become precipitated or cloudy, the column become high presure very soon, it seems the particle is so small, it pass the filter and went into the column, and so far, I didn't find any way to regenerate the coulmn. Any body can advice what is the mechenic for this solution to precipitate? any method to prevent this? any method to regenerate the column? Thanks.

Ammonium acetate would not be my first choice at pH 6.7. Acetate has a pKa of 4.8, and ammonia has a pKa of 9.2.

That said, I would not expect ammonium acetate to precipitate, but I would expect it to make a nice nutrient for microorganisms. You should be able to prevent bugs from growing (or at least slow them down) by adding 5 - 10% organic solvent (e.g., methanol) and by preparing fresh buffer every day. A plugged column can sometimes be regenerated by back-flushing (reverse-flow), but check with the column manufacturer first. Not all columns can be backflushed.

first u use mobile phase after filter it with 0.2µm filter. then with organic modifier. i dont think this buffer would be pecipate . i also want to know what is your column particle size. if this is 3µm or less then the column back pressure is also increased.

hi, thanks for reply. However what I saw is precipate instead of microorganism. After only a few days, I can see the whrite powder like stuff at the bottom of the bottle, occasionally the whole solution become cloudy. Some time take longer, some time take shorter. Please advise

I have routinely used 20 mM ammonium acetate buffers for various methods I have validated. Never once have I seen this buffer precipitate out of solution on me. I have also not seen it grow organisms up to one month out (it might even be longer; I only evaluated mobile phase stability for 1 month).

As suggested, I would not choose Ammonium Acetate for a buffer at pH 6.7. For this pH I would look at dibasic ammonium phosphate, dibasic potassium phosphate, and tribasic sodium citrate (be careful of stainless steel parts on the HPLC, this will corrode them).

Try giving your glassware / pipettes a good rinse before using them.

Is your acetate solution aqueous, or does it have an organic component?. I'd suspect contamination ( CO2? ) or evaporation.
I assume all your reagents are HPLC grade, and any filtration system is really clean, if not your problem could be an impurity/cotamination.

If you do have to use it, try putting some in the fridge, and filter cold. maybe the warm solution will not precipitate so fast.

Please keep having fun,

Bruce Hamilton

Perhaps the precipitation is due to the preparation of the mp. I never see this problem with my prepared solutions.

However, a newbie in our lab made the Ammonium Acetate buffer, and then he added this buffer to pure Acetonitrile (rather than the other way around). It did not dissolve nicely. He got a lot of white chunks in the membrance filter, and the mp did not work!
As a corrective action, I had to give him 2 rule-of-thumb:
1. Always add organic solvent to aq. salts.
2. Mix/stir for >= 1 min for each 100mL prepared. So, 1L of mp will need >=10min of mixing. (Our newbie mixed approx. 1-2min for his solutions!)

Hope that this is useful, and Merry Christmas.

Alfred

I do use ammonium acetate buffers (10mmol/L, pH=8.5) on occasion and they are very good growth media. They usually become hazy after about 5-10 days no matter how clean the bottles were.
It is also interesting to point that the growth of microorganisms can very significantly from one lab to the other.
I remember using triethylammonium acetate buffers (100mmol/L, pH=7) for weeks with absolutely no problem while others reported growth within a few days.

Anybody eve use ammonium acetate beffer as one of the mobile phase solvent? I use it in one of my method, it is pH 6.7 ammonim acetate, 0.02M. I found this beffer or solution precipitate from time to time, after the solution become precipitated or cloudy, the column become high presure very soon, it seems the particle is so small, it pass the filter and went into the column, and so far, I didn't find any way to regenerate the coulmn. Any body can advice what is the mechenic for this solution to precipitate? any method to prevent this? any method to regenerate the column? Thanks.

from your statement i infer that you have an inline filter through which your buffer passes. if this is the case then you may have a clogged filter rather than the column.

i have experienced clogged inline filters and the pressure rise that occurs. it seems as though the problem is the column but nothing you do to the column will remedy the problem. then you clean or change the inline filter and everything goes back to normal.

in the future, you might want to reconsider sending a cloudy mobile phase through your system.

Hi, THANK ALL FOR your input.

re: mdfenko;

It is amazing that my filter is fine, it seems that the particle is so small that it can pass through the filter no problem. We have another method using ammonium acetate buufer, which has the same problem. but it didn't damege the column.

re: shaun78

Hi, I don't understand why ammonium acetate has phosphate?


Re: Alfred88

I didn't mix it with ACN, in fact my supervisor suggest me to premix them to avoid the possible precipitation/microorganism.


Since in my lab we have two method using this buffer, quite some chemist ran these methods, we all obersve this phenomena for this buffer from time to time, we have really hard time to explain this. And I feel amazed that not so many people have this problem. welcome more suggestions!

Emily,

shaun78 was pointing out that ammonium acetate does not have any significant buffer capacity at pH=6.7 (pKa of acetic acid is 4.8 and of ammonium is 9.2 just like Tom pointed out above)
I am pretty sure that you experience microbial growth. As your supervisor (and Tom again) suggested, addition of acetonitrile or methanol (5 to 20%) will suppress that. Frequent preparation of new buffer also helps.
Microorganisms can pass inline filters (depends also on the porosity) but will usually clog the column.