Chromatography Forum

As most of us know, an aqueous mobile phase solvent buffered at difference pH levels is often used for gradient elution in RP-HPLC. Since 1) bacterial grow is a major problem affecting integrity of aqueous mobile phase, and 2) growth rate of bacteria is highly pH-dependent, logically, (true) expiration of an aqueous mobile phase buffer should be related to its pH value. I wonder if some of our fellow chromatographers here have more experience on this and would be willing to share?

Many thanks in advance.

In my experience, the rate at which bacteria grow in a buffer depends on other factors as well. I've seen bacterial contamination show up in very acidic buffers in a very short amount of time. A lot depends on the buffer salt. I don't know exact numbers, but I'm pretty sure a phosphate or bicarbonate buffer is going to go bad a lot quicker than a borate buffer, due to the preservative effects of borate salts. Temperature is another big factor. In my lab we add sodium azide whenever possible (ie, when it won't interfere with a reaction to be performed later.). Sodium azide is excellent at preserving near neutral aqueous buffers.

And if you do a lot of work with the same kinds of buffers, the lifetime will gradually get shorter as you do selective breeding for bugs which "like" those buffers!

This is another good reason to pre-mix solvents. If your gradient method starts at 95%A and 5%B, rather than using a gradient pump to achieve this just pre-blend your A solvent to that condition and adjust your gradient settings or B solvent to compensate.

This should help stave off bacterial growth and also gives you lower noise in your chromatography.

Scott / Tom / Peter: Thanks indeed for sharing your valuable insights.

Bacterial growth in a phosphate buffers - permanent headache.

Usually we prepare the minimal volume buffer ,and should not store its remainder.

just a related question really... does anyone know what the pinkish stuff is that "grows" in demineralised water supplies? Is it specific to our lab, and is it mineral or vegetable? I'm amazed anything can grow with no nutrients, but I wouldn't put anything past nature.

There is no microbe growth without nutrients, pink sounds definately like microbes.

http://www.sepscience.com/emails/HPLCSol65NA.pdf

Article from John Dolan on mobile phase expiration...

@lmh
Probably Serratia marscens (spelling?). It's the pinkish stuff usually found in bathroom crevices, tiles, toilets, etc.

There is no microbe growth without nutrients, pink sounds definately like microbes.

Pink sounds like girl microbes.

CPG, if only there were the equivalent of a 'rimshot' for jokes on the Forum....

I guess that adding something like 5-10% of methanol would be very helpful to increase the time you can hold on to your mobile phase. Of course provided that this will not affect your chromatography.

Thanks, all, for helpful and/or amusing reflections on pink stuff in our lab. I will feel differently towards it next time I see it growing.

We never store water and aqueous buffer mobile phase more than one day. If it is a mixture with organic solvent(s), we store it in the refrigerator to avoid evaporation than may change the composition of the mixture, at least for 6 months.