Has your HPLC ever been infected?

[Jade.Barker]

Can anyone share a story about a system infection? I'm happy to say we have not had one (yet). But some of our operators do not seem to take the precautions needed to prevent an infection very seriously. Maybe a couple "scary" stories would help?

I am specifically interested in these talking points:
1. How did it happen?
2. How long were you off-line?
3. What efforts were needed to fix it?
4. What was the approximate cost of lost productivity?

[ACPilot]

infected with what? are you talking about a biological infection or a computer based infection?

[Jade.Barker]

Sorry for the Confusion: Biological infection

Here's the story: Since we mix our own mobile phase (non-sterile) it is important that we change the mobile twice a week. I explained the problems a microbial infection can cause. I thought they understood - but evidently not...

I had a group of old mobile bottles waiting to be neutralized, dumped and washed. Each one had a small amount of mobile left. I came in this morning and saw the instrument resevoir was suspiciously higher than expected. This was extra strange because that was the last "fresh" bottle... Then I saw the empty bottles. Some one grabbed about 6 mostly empty bottles off the counter and poured them all into the resevoir bottle.

The best/worst part? There was NO need to add mobile, I had turned the flow off overnight (very easy to check on our system). It was lucky that none of the "dirty" mobile went in the system. But I need to talk to the team, this should never happen. Those bottles could have been neutralized already, or used for something else...

Why they didn't make fresh mobile? Even if they were super busy, they could have just checked the flow and shut it down to zero if they thought it might run dry. Adding whatever liquid you find on the bench? NOT Okay. I need this concept to sink in.

[skunked_once]

I respectfully suggest a demonstration that may help them see the light. Save the dregs of several individual coffee cups for a few days then when it's coffee time just top off their cups with the leftovers That should get their attention!!!

[Bruce Hamilton]

I've used wildlife-filled phosphate buffers because I misread the expiry date. The solvent reservoir filter blocked first, the pump flow became intermittent, and the pump inlet frits also partially blocked.

The system was easily cleaned, so time lost was only a few hours. Now I read labels a little more carefully most of the time.

The wildlife growth rate can be quite impressive in neutral phosphate solutions, so I'd put some well-labelled bottles in a constantly-warm area and wait a few weeks. Seeing mould colonies swirling around is a good lesson.

Please keep having fun,

Bruce Hamilton

[Uwe Neue]

We should keep in mind though that wildlife proliferates in water at pH 7, but not at pH 2 and not when 10% acetonitrile are added.

[khw]

I also learned the very hard way that some bacteria like sodium azide and grow there. It took three customer service reps until they found evidence of life on the spheres in the valve that caused sudden pressure drops. Since we premix our water with MeOH or ACN, this did not happen again.

[danko]

Uwe
We should keep in mind though that wildlife proliferates in water at pH 7, but not at pH 2 and not when 10% acetonitrile are added.

K.H.W.
Since we premix our water with MeOH or ACN, this did not happen again.

Yet another reason for pre-mixing – and there are many more.
I would only add that high salt concentrations act as a preserver as well. Typically in the case of ion- exchange and HIC – the highly concentrated eluents anyway.

Best Regards

[Jade.Barker]

Thank you for the input everyone!

I definitly like the idea of making swamp coffee but I'm too nice to put that into motion... However, I think I will put some mobile to grow for a couple weeks. I better hide it though, or they might be tempted to use it in desperation.

We are doing Ionic separation we use a water + 0.5 mM H2SO4 mobile (pH is around 3.3). I know we have microbes in the lab that will grow at that pH because of the corn fermentation samples we bring in. They manipulate the pH in the plants for enzyme activity, but they still can get a nasty infection. We do HPLC and Petrifilms. Most of the samples we get are growing something they shouldn't... but then again thats why they send them to us.

Anyhow, Our researcher had been using 5.0 mM H2SO4 (at his old lab) and he said it will *definitely* grow things. I guess some of our partners in South America are adding a preservative to their mobile phase - but I haven't discussed it in depth. I guess it is more popular down there?

[mbicking]

So, you're looking for horror stories? I've seen a few.

My list is topped by the ethanol fermentation industry as noted earlier. The standard method uses 0.05 M H2SO4 and an ion exchange column with RI detection (ugly and slow!).

One lab was having continuing column plugging problems (in an earlier visit I had explained the value of filtering, so that wasn't the problem). I ignored the fact that there were huge colonies of fuzzy fungi (or whatever it was) in the waste bottle; this was the least of their problems. Actually, they seemed to be doing most things correct - changing solution regularly, cleaning the bottles between changes, etc., but they still were having problems. But when I looked at the inside of the reservoir cap, there was a ring of black slime. There were bugs growing in/on the cap. Every time they changed the bottle with clean mobile phase, the re-innoculated the solution when they screwed on the cap. Now, the capy was easy to clean, but the LC ...

Another ethanol plant (my first visit), hadn't learned the value of filtering yet. And they seemed to think that the third shift people would find this to be either too difficult or too time-consuming. The entire instrument was a microbiology experiment!

In both cases I used a 10% bleach solution to clean the lines, the degasser, and the pump. It was a little over the top, but I was so disgusted with how the instrument was being abused that I responded in kind! When we looked under the microscope at the visible stuff coming out of the degasser, we saw a nice collection of fungal mycelia and other "things." Remarkably, both instruments came back fine; it's a testament to their ruggedness.

Each event only took a few hours to fix, but it was a full day before the systems were up and running again.

[Kostas Petritis]

I have witnessed in the past an Agilent 1050 pump which aqueous lines had developed colonies (black matter here and there attached to the inside of the tube and moving with every pump stroke). The humidity inside the lab was higher than you would expect which promoted microbial growth. The Agilent (then HP) service engineer almost had a heart attack when he came to repair the system.

The people running the instrument knew nothing about HPLC back then, trying to separate analytes by using a SCX column and a gradient from water to ACN without any additives added... and wondering why it does not work...

[ryan]

I have had this problem with the agilent 1200, and lost a few columns that became irreversibly blocked. The method we were stuck with was with phosphate buffer gradient with AcN on seperate lines, but the guys who developed it insisted there was no problem with bacterial growth. We changed the buffer daily but still got the problem repeatedly. WE had to flush the system out with acid, disinfect all the bottles and lids to clean it out. Lost several days of work that way. In the end, we just changed the method to have a little bit of AcN in the buffer solution and then adjusted the gradient accordingly.

[Jade.Barker]

The standard method uses 0.05 M H2SO4 and an ion exchange column with RI detection (ugly and slow!)...

We are doing this too. Except 0.5 vs. 0.05. I think we may be stuck with this method, since our system is supposed to "represenative" of what the plants are doing. Basically, if our HPLC numbers are too different from the plants, they get suspicious.

But when I looked at the inside of the reservoir cap, there was a ring of black slime.

Wow, that nasty. This makes me feel a little better about our lab. Also, I had been feeling a little lazy, since I never drilled the caps for the nalgene bottles we use. We cover the inlet with petri film. Sounds like this time the "easy" way was the good way.

So, if I see floaty bits in the waste water it's bad? We use a 4L jug that just sits until it is full. I had assumed the growth was happening after the fluid hit the bottle. I hope this isn't some kind of early warning I should be watching for...

[mbicking]

The bugs in the waste bottle are technically not a problem, as long as those solutions remain downstream of the instrument. However, this does indicate that your solution can serve as a growth medium, and your environment is providing the organisms - beware!