hello everyone,
As a beginer in the field, I am always wondering how to properly prepare and store my buffered mobile phase, say MeOH/H2O (80/20 by volume), with 20 mM potassium phosphate. I have heard that bacteria can grow in phosphate buffer. How would I prevent this from happening? I usually prepare 500 mL or 1 liter mobile phase, which will last me about a week. I always keep my mobile phase in the refrigerator when not in use. Is that good enough? How fast will the bacteria grow? Thanks in advance.

In my opinion buffered mobile phase which is being refrigerated should last at most 5 days. The fact that yours has such a high content of organic would also support this. My advice is that unless your experiencing problems keep doing as you are.

Hope this helps
Adrian

I agree with Adrian, your mobile phase has a high content of methanol, which prevents microbial growth. I'd only add that some experienced people have advised me not to prepare much mobile phase because of volatility of solvents; composition can change and this would affect separation. They say to prepare only the quantity that I'm going to use in a day. I'm not sure how much solvents can evaporate, but that's the tip.

Good luck!

Thanks for the tips. Actually I had the concern about solvent volality. However, I also worry about the reproducibility of the mobile phase composition, especially when buffer is involved, issues such as pH, ionic strength etc. Any comments?

Make sure that you keep good notes about how you prep your buffer, including any mistakes, so you can repeat your preparation each time.
I also use a 20mM phosphate buffer, and I filter it EVERY time I use it. (I don't mix my organic & aqueous, so mine grows "stuff" in a day or two).
Hope this helps!
Lisa

In general, 10% or so of methanol *should* be bacteriostatic, *but* . . . let me tell you a story:

A decade and a half ago, I was doing a lot of single-column ion chromatography, using phthalate buffers. We found that the buffers had to be made up fresh every day. Following up on a paper published by the person who had originally developed the technique we were using, we switched to p-hydroxybenzoate buffers. and were *amazed* at the difference. We could use a batch of buffer for a week before bug growth got to be a problem.

A few months later that person visited our lab and happened to mention that he had recently switched from p-hydroxybenzoate to phthalate because he found that he had a lot fewer bugs growing in that buffer.

The point? We concluded that we had been separately doing selective breeding experiments on the local microfauna; basically, the survivors in each lab were the bugs who could tolerate (if not actually thrive!) in the buffer being used.

I've talked to a few people recently who have commented on bug growth in mobile phases with > 10% organic, so the old generalizations may no longer be valid.

-- Tom Jupille / LC Resources