sodium azide question

[Ron]

I have also had experience with a couple of episodes of bacteria growing where it should be impossible. One instance was due to a technician only hearing the clean portion of clean and sterilize before using the glassware instructions. Since bacteria grow in the solvents, filtering through a membrane filter can be a simple and useful precaution.

[DR]

High background absorbance - but that has to come from a larger colony, and there's no room for that *in* a system, only in the jugs.

[SSGG]

How about a bacterial colony in the column?

The reason I ask this is that I have seen the same very broad peak (max at 205nm) eluting from all of my RP columns, even after thorough flushing (and storing in 70 - 100% ACN).

I have always assumed that it is an impurity from my sample, maybe a very strongly retained protein that I have not managed to flush out..

I have not managed to get rid of it completely. It's still stubbornly lurking in my columns after every analysis. So far, I've only been successful at reducing the peak area (after prolonged cleaning with H2O/ACN gradients).

Any ideas?

[DR]

Does this broad peak elute only during gradient runs?

[syx]

How could ‘bacterial particles’ be eluted by mobile phase through the column? I think they tend to clog the column by their insoluble bodies than to create a band.

[SSGG]

Dear DR and syx,

The broad peak, which I don't know whether is due to carryover from my sample, or something else entirely, elutes in both isocratic and gradient runs.

However, during gradient runs (increasing ACN), it elutes earlier.

Presumably if bacteria can cause high background absorbance, as DR said, the stuff the bacteria give off (metabolites? degradation products?) can absorb some UV.