Chromatography Forum

Hi guys,

A friend of mine developed a HPLC method with column conditions at neutral pH. The mobile phase buffered with phosphate at pH 7.2.

I am a little concerned this might yield bacterial growth pretty rapidly and can affect chromatography.

Let me know, if this reason of mine is plausible or am I just getting too worried.

Yes, bacterial growth is a possibility, especially if there is no organic solvent present, but I doubt it would be catastrophic. I would certainly prepare the buffer fresh daily, throw away the buffer at the end of the day (if you are not running overnight) and clean the reservoir thoroughly.

Hi Mohan

As Tom mentions above, if an adequate fraction of the mobile phase is some kind of organic solvent (e.g. ACN, methanol, Iso-propanol etc.) it will effectively prevent bacterial growth.
Also, if the buffer is concentrated (e.g. > 0.1 M) it will prevent growth as well. So maybe your friend knows what he/she is doing, after all.

Over the time, I’ve developed quite a number of methods using mobile phases at pH around the neutral, with no or little organic modifier in them (primarily HIC, IEx and SEC) and I never saw bacteria choosing my eluents for their home. Maybe they were clever enough to realize that it wouldn’t be in their interest if they let somebody see them. I don’t know
Even the low concentrated eluents survived several weeks’ idleness. As for the high concentrated; I have the feeling they can “liveâ€

"I am a little concerned this might yield bacterial growth pretty rapidly and can affect chromatography. " - if you use fresh buffers then you are getting too worried

As long as you prepare fresh buffer for analysis as Tom suggested you are safe

It doesn´t do any good to prepare fresh buffers if your water is alive.
Apparently Dancho has a lab atmosphere and the skills to work cleanly. Cleanliness, all around, goes a long way in saving time in this. For instance, I prepared several different phosphate buffers more than two years ago and opened the original volumetric flasks only 2 or 3 times since then (kept in the cold room when not used): Not one spec or other sign of any microbes.

" I prepared several different phosphate buffers more than two years ago" - being busy, huh?

Thanks guys,

Apparently, the method can be developed at a lower pH. My friend chose a higher pH for some reason.

I just wanted to make sure that no strange ghost peaks appear later on - as it is a long end study.

I have done method development at pH 7 since 1994 without problems.