Preventing bacterial growth

[mtorrey]

Seeking advice on preventing bacterial growth in aqueas mobil phase for UPLC/MS/MS. Solvent A is 5 mM Ammonium Acetate, ph 10. Solvent B is ACN (100%). Performing 30:70 A:B isocratic inlet method.

I've been adding ACN (1%) but sources from Waters say to use 10% organic. I don't want to use that much. Haven't seen any contamination yet but wonder if I'm adding enough ACN to prevent bacterial growth.

TIA
Mike

[southp]

Is there a reason for not wanting to use 10% ACN as your A solvent? It seems like you could prepare A with 10% ACN and then set the mixing to 33:67 A:B to achieve approximately the current 30:70 isocratic composition.

Or you could pre-mix the 30:70 in a bottle and be protected from bacteria/algae growth that way. I was just at a UPLC demo, though, and Waters claimed it's more accurate and reproducible to let the UPLC do the mixing for you.

[Dan]

Mike,

That 10% organic is the rule of thumb that we probably have all heard.

It is possibly that you can get by with less than 10% organic to inhibit bacterial growth, it depends on the situation (buffer type, concentration storage, etc.). You would have to be (or consult with) a microbiologist.

Some buffers are better for long term storage than others. Phosphate buffers at around pH 6-7 will show bacterial growth within a few days.

I think that buffers at high pH are less susceptible to growth than those at lower pH. I don't remember for sure. Any micro people out there?

Regards,
Dan

[mtorrey]

It seems like you could prepare A with 10% ACN and then set the mixing to 33:67 A:B to achieve approximately the current 30:70 isocratic composition.

Thanks for the idea, I'll try that.

Mike

[Uwe Neue]

Like with everything else, simple rules are not capable to deal with more complex situations.

Keep in mind that you want to use a mobile phase that does not promote the growth of bugs. pH 7 phosphate buffer is a rather good medium for bug growth. If you make this pH 2, or pH 12, close to nothing will grow in your mobile phase. Similarly, adding 10% of acetonitrile to a pH 7 phosphate buffer prevents bug growth.

In a bicarbonate buffer at pH 10, you may still get away without a chance of bug growth, but maybe you need to add 2% of acetonitrile. Same question at pH 4.5 in an acetate buffer.

Even if you are working with pH 7 phosphate in 100% water, you can prevent the growth of undesirable entities by frequently replacing the contents of the container.

[DR]

Premixing will result in a smoother baseline anyway. Having 100% Aq. A and 100% organic B is really convenient for development, but it's no way to run an isocratic method, particularly if you were using a high pressure mixing system.

[james little]

we use 3% acetonitrile in 97% water containing 2.5 mmolar ammonium acetate routinely for LC-MS. Solvent B is pure acetonitrile.

Add 25 mmolar ammonium acetate in methanol (0.1 ml/min mixed post column with 1-1.5 ml/min eluent). The post column addition allows better ionization at 100% organic.

No problems with bacteria growth over the last few years.

Before we added 3% acetonitrile, had a real bad problem with filter plugging from bacteria.