Chromatography Forum

We are analyzing samples containing high amount of nutrients, which are sent to us after filtered in another site. We just notice that new micro organism is growing in the samples and eventually clog our needle/tubing/columns. Since we cann't immediately change the method by adding a filtration step we are wondering if there is a way to clean up the system after the system is contaminated by the micro organism. ACN and nitric acid will kill them but will any of them is able to digest/dissolve the dead body and wash them off? or we have to replace the parts?

Thanks
Jim

A cleaning procedure for the UPLC employing inorganic acids (phosphoric acid or nitric acid) should indeed remove all organic contamination - including dead or alive organisms. Make sure to do it properly, follow the procedures of your instrument manufacturer and work safely.

These procedures are obviously used with the column and the detector disconnected. For your column, you can try column cleaning procedures or just use a new one.

Dear RnDirk,

Thanks a lot taking time to answer my question. May I confirm with you that what you answer is what your personal experience/general practice or just 2nd hand knowledge. I am sorry this sounds distrust but you know change in our industry has big impacts at large scale. I rather make sure 100% that it is good and then implement than proceed without good confirmation. Thank you again.

And hope someone else can confirm this.

To confirm part of RnDirk's response:

Our columns are frequently used with salt buffers in pH ranges hospitable to microbial growth (ion-exchange; HIC; SEC), so we are confronted with this problem frequently. In our experience, once an HPLC column has been contaminated by microbial growth, the resulting increase in backpressure is irreversible and it should be discarded.

People who use only reversed-phase HPLC may not encounter this situation, since the mobile phases are frequently inhospitable to microbes.

Thanks Andy.

I resonate your take, that it is difficult to remove live/dead microbial out of the LC system once the system is contaminated by microbial. Neither phosphoric acid nor nitric acid is able to dissolve or digest them into small enough. But this is just my guess. I like to know if someone has tried this cleaning and know it works or not.

Actually, the situation is more nuanced than that. There are solvents that will reliably dissolve bacterial cell walls, fungal spores, and the like. However, any such solvent that I'm familiar with will attack the coating on an HPLC material before it will dissolve the microbial detritus. That's why you remove the column before treating the HPLC system to sterilize it, and why you should consider throwing out a contaminated column.

Hi Andy,

How much do I need to pay to get the secret and powerful solvents name/recipe, which are capable of "reliably dissolve bacterial cell walls, fungal spores, and the like". You don't need to take any liability and I will take full responsibility of any consequences due to using the recipe.

Thanks and have a great weekend,

A blend of hexafluoro-2-propanol and concentrated formic acid, 8:2, will afford a clear solution in minutes at room temperature even if the sample is a bacterial cell wall pellet (which has a solubility comparable to that of nylon). The alternative recommendations of 3 N nitric acid and the like will serve to sterilize the HPLC system, preventing further contamination for a while. The nitric acid treatment will also passivate the HPLC system's stainless steel components for up to 3 weeks, rendering them resistant to attack by chloride ion.

THANKS!!! Andy

Two additional questions. When you said "clear solution" did you refer to real solution (means that cellulose becomes smaller than small oligosaccharides) or only the particles are too small to bee seen by eyes?

when you said the alternative is 3N nitric acid did you mean that 3 N Nitric acid also dissolve cell wall into a real solution (particles below 0.1 um)?

I appreciate your expertise and helps!

I meant that the cell wall polymers (including the ones that are protein-based as well as cellulose) are indeed in solution, not just a colloidal suspension. It's a nice way to dissolve membrane proteins that won't go into solution otherwise. We've used this method to isolate pathogenic prion proteins from brain tissue. The prions went into solution 100x slower than anything else, making it easy to isolate them via centrifugation into a pellet.

Regarding the ability of 3N nitric acid to dissolve bacterial cell walls and fungal spores: I defer to the members of the forum with more experience than me with this solvent.

Dear Andy!

You are the MAN! You made my day! Thanks a lot!

Jim

The accolade is nice. However, before you fill your HPLC system with this solvent, better make sure it won't dissolve your pump seals.

Andy, thanks again. Do or did you use this solvents to clean miroorganism contaminated LC systems in your lab or do you know any lab use this cleaning as a routine practice?

I have used 30% hydrogen peroxide before when we had really bad algal growth in an HPLC system, followed by water then 5% nitric acid then lots and lots of water. Peroxide and Nitric do a good job of oxidizing organics, so it is best to do it before you change the pump seals not right after.

If you have Pyrex, Kimax, or Duran solvent bottles, you can put them into a muffle furnace at 400-450C for an hour and get them much cleaner than just washing with soap and water.

Thanks James.

It seems that it does need to change the seal after the peroxide and Nitric treatment. Correct?

Jim