Chromatography Forum

In our laboratory, we have been working with 2 UPLC systems for about 2 years now.

Since June 2006, we observed problems of overpressure on columns after about 150 injections. We submitted the problem to the vendor (Waters).
They replied that it was certainly due to the development of bacterias in the column. I put Water 0,2 µ in A1 and acn 0,2 µ in B1.

Recently, I've put Water 90 / acn 10 in A1 and then the problems of
overpressures seems to disappear.

Does anybody already had this problem ?
What was your solution ? What's your experience?

Thanks for your advice

Pierre

If the problem has gone away, what's the problem? (I'm not being sarcastic here, just trying to ascertain the true question).

Hi pve,

If I read your post correctly, you have in A1 pure water (0.2 um filtered)? I don't think that's a good idea since other microorganisms (quite apart from bacteria, such as molds & fungi) can also enter into the vessel and start multiplying. I would actually have some organics in it (ie, premix and filter your solvent before use) to discourage or slow down microbial growth.

In our laboratory, we have been working with 2 UPLC systems for about 2 years now.

Since June 2006, we observed problems of overpressure on columns after about 150 injections. We submitted the problem to the vendor (Waters). They replied that it was certainly due to the development of bacterias in the column.

One question, if it is microorganisms, why didn't you have the problem before Jun 2006?. What changed in June, your water system?. The recent issue would appear to be solved by adding acetonitrile to the water, so you could keep doing that.....

It appears that the column storage solvents were OK, so if you want to change back to 100% water, work on your mobile phases and samples.

If, like me, you prefer to use 100% water, you need to change it frequently ( daily ), and ensure that the final filter of the water system is regularly changed, and well flushed before dispensing. Ensure that nothing is left in contact with static water ( reservoirs, degassers, etc ) for more than 24 hours.

Bruce Hamilton

Dear Friend,

When ever either in HPLC or UPLC do not keep 100% water and if so required try to change every day. Generelly we use to keep 90% water and 10% organic phase and the same will be used for column washing and in other hand 90% organic phase and 10% water before flushing the column with 100% organic phase for storage of column.

If any water remailns in column it will help to develop bacteria and other microorganism development. so when ever you are using water mix with 10% organic phase so that you wont phase such problems again

g.r.reddy.

We are also assuming here that suppliers of water fill and seal their bottles properly. Do they seal with an inert gas in the headspace or some other method? Do they use a stabiliser?
I know that we filter prior to use anyway.
WK

Two semi-related questions for you:

1. Is running with 0.05 % v/v TFA or similar pH change in pure de-ionised water enough to prevent excessive microbial growth and the bad baselines that result?

2. I heard from a colleague in Pharma R&D that UPLC had poor baselines cause by the high pressures and he was struggling to see distinguish the 0.05/0.03 % area peaks above the baselinse noise. Anyone else had this particular issue?

Regards, Pat.