chromatography at neutral pH (around 7.2)

[mohan_2008]

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.

[tom jupille]

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.

[danko]

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€

[grzesiek]

"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

[HW Mueller]

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.

[grzesiek]

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

[mohan_2008]

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.

[Uwe Neue]

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