HPLC unknown contamination

[Uwe Neue]

If you mix water and organic online, you can get bacterial growth in your water (reservoir, line etc.). Washing with acid will kill the bacteria, but you need to figure out how to prevent this from happneing in the future. In your case, you seem to run isocratically. Premixing the mobile phase would solve the problem.

[ym3142]

Dear Jim B,

While we understand you are very frustrated and you had tried everything I am not clear of what you did. Uwe did guess you were running isocratic. But why you were shifting from ACN to water?

What are you intending to say: "It runs at the solvent front and it's very strong absorbance."? are you saying you saw the peaks when mobile changed from H2O to ACN? If it is, is that not normal for UV detector?

of course you also indicated the peak at about 30% ACN. just confusing for me.

[unmgvar]

1050 old work horse?
do you have an old degaser as well that comes along with it, with about 50 ml of tubing that simply loves to get infected after being so old.

check the material but if memory serve me right you should be able to flush it with a strong organic solvent, even by hand with a syringe.

on a dionex system we even used a little a low % detergent solution overnight to clean the lines
this is a procedure we used for the lines of the on-line dissolution every month

[carls]

Hello saioa,

Were you able to resolve your contamination problem? I am having a similar one and it's very frustrating. I'm seeing a peak in my no injection runs near the end of my gradient (at 100% ACN) and I know from looking at the MRM transitions it is a few benzodiazepines in my method (oxazepam, temazepam, and lorazepam) causing this peak. I have run the three blank gradient test and the response increases with increasing equilibration time. My aqueous mobile phase is 0.1% acetic acid and I have tried two different bottles of acetic acid, flushing the line with IPA, trying formic acid, using the other line we use for our aqueous solvents, and preparing the mobile phase from two different sources of water and the peak is still there. I am at a loss. Is there anything that worked for you? Any help you can provide would be greatly appreciated.

What HPLC system are you using? Are you running an injector program or otherwise moving the inject valve just prior to the appearance of these peaks? if so, sounds like the injection valve needs to be cleaned and rebuilt.

[carls]

Hi, I too have been having a problems with contamination. I keep getting two peaks with absorbance maximums at 245 and 250 nm respectively that elute at about 30% ACN/water from C18 column. The problem is not in the column or ALS. I have tried changing A and B solvents about six times, removing the sinker frits, everything I can think of. We have also cleaned the pump head and changed the seals (it's an old workhorse HP 1050). And we ran a liter of IPA/MeOH/water 1:1:1 through the system over the weekend.

Running solvent with no column, I can see the 245 and 250 peaks when I shift from 100% water to 100% ACN. It runs at the solvent front and it's very strong absorbance. But there is nothing in line, no column, no injector, so where is the chromophore accumulating? If I run 50/50, I can see some aborbance at 250 nm in the PDA scan. If I zero balance the detector at 100% ACN the absorbances I see at 100% water are: 220 nm = 0 mAu, 254 nm = 8 mAu, 280 nm = 8 mAu.

I suppost after all this it can still be the water but I have tried milliQ water (18.2 MOhm) fresh off of the purification system, as well as two bottles of freshly opened HPLC water. I just can't track it.

I will try the nitric acid wash next.

FYI, I don't run exquisitly sensitive experiments so these are not small peaks, but huge ones. They just appeared out of nowhere about a week ago. Any newsuggestions are welcome.

As has been suggested, try running your experiments with the degasser bypassed.