Chromatography Forum

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

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.

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

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

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.

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.

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.