The main problem you experienced was probably caused by a lack of flushing the HPLC system down at the end of each day. This system is not designed to be left in PBS overnight. The salt content will build up inside the Teflon membranes within the degasser and also in the rest of the HPLC system and result in contamination. It may also contaminate the multi-channel-gradient-valve (MCGV) used with quaternary pumps too, causing problems with other channels. Regular flushing of all of the lines, each day, with DH20 and then some organic added (e.g 20% MeOH) will clean out the system. Also, because you are using a model G1322A degasser with very large 12 ml volume vacuum chambers, you need to take this into account when flushing EACH channel. The low pressure connection tubing adds another 5-8 mls of volume to each channel, so you really need to flush about 60mls of wash solution (3x, because you actually need ~20 mls total for one flush, per channel) through EACH channel at the end of the day (and also when you start the system up each day). This is required to maintain and operate the HPLC system (Part of normal HPLC maintenance/operation). Overnight, gas bleeds back into the Teflon low pressure tubing and these bubbles need to be flushed out before you can start equilibrating the column. Open up the prime purge valve to waste and flush down each channel, one-at-a-time, 100% A-B-C-D. This is something that I teach in all of my intro HPLC classes. Doing this will save you a lot of time and help maintain the machine in a clean, bubble free state.  BTW: if you have an HP/Agilent Quat pump, be sure and set the max pressure to

I think your idea would do the trick.  You could add pepsin to the hydrochloric acid solution to remove any protein as well.  

Hi Karen,

thanks a lot for sharing that information with me. After 2 L of HCl dripping slowly over night, I put the degasser on pepsin/HCl and incubate for several hours. We want to disassemble the degasser later on, maybe it is possible to isolate the solvent reservoirs and clean them mechanically.

I'll post some images to have this question as a kind of reference "blog" for others.

Pump through IPA before you go back to your aqueous mobile phase.

When you open up the degasser there is a big plastic block, which cannot be further disassembled without risking hardware damage. There is not much point opening it up in your case.  You can try hooking up a peristaltic pump and run acid/base/IPA, maybe with some surfactant, through the system at 5mL/min.  Heating the liquid being passed through to 50C can also help.

I guess what you see is a precipitate of the buffer. How long do you store it and what is the temperature ?

You may know this, but Agilent has extensive video and animated documentation online for servicing HPLC parts.  This current problem may be solved already, but you may want to refer to their online documentation for other issues. 

I'm not sure if you have/had bacteria or salt precipitates, but the ideas above seem like good answers to me. 

Hi All,

thanks a lot to everyone who contributed to my question! I think we solved the problem but still don't know whether it was bacterial contamination or salt precipitate. Referring to the question of Joachim: we "stored" the solvent line at any time in PBS at room temperature, at pH 7.4, a lot stuff can grow there... now we will exchange the PBS buffer after usage with 0.02% NaN3 in water to remove buffer and salt and keep it toxic for bacteria. With this, the problem will most probably not occur in the future. We never had problems with the other solvent lines (A1= 5% AcN/Water 0.01% HCOOH, B1= 95% AcN/Water 0.01% HCOOH and B2 = 0.02% NaN3 in water). Thanks to James, I already checked the Agilent site before I started any cleaning procedure, but there was nothing to find concerning my issue. James is right, I also strongly recommend the Agilent videos and documentation to everybody working with Agilent instruments! It really helped me very often in the past (unfortunately not this time...).

Because I promised to send pictures to have this question as a kind of "reference blog" for everybody, there you go (images from disassembling the degasser). In the next post I will illustrate the details of the steps performed. Beforehand I can tell you that it is not necessary to disassemble the degasser if you have problems as I had, although it is possible and was a nice experience :-)

These steps were performed for G1322A Degasser decontamination (all steps at room temperature):

detach degasser outlet from maifold plate of the pump unit

change solvent to 0.1 M HCl (2L), let drip by gravity over night

Change to pepsin/HCl (approx. 30 ml for flushing, degasser volume for each solvent line approx 10 ml,) and incubate over night

flush with HCl and then water afterwards. We tried also iPrOH.

this did not help very much (by applying vacuum with peleus-ball there were still some clumps coming out), so we decided to disassamble the degasser (which is not necessary).

The degasser pretty much consist of a vacuum chamber, a small vacuum pump and a valve. The solvent line from the liquid tanks is then splitted into many smaller tubings (I guess permeable for gas) to ensure maximum surface area (I call them "solvent splitters" which may be wrong, sorry for that). While putting vacuum in the chamber, gas is released from the system. For all solvent lines, the total volume of the smaller tubing is approx. 10 ml.

We installed an external peristaltic pump (I did not want to use HPLC pump, which may be contaminated then) to the solvent splitters and purged with 0.1 M HCl first @ 10 ml/min for 20 min, then 15 ml/min for 20 min and raised the flow to 50 ml/min at the end. This massively pushed out clumps and white stuff. The pressure from the flow matters!

The degasser outlet tubing was still dirty and could be physically cleaned with an old green Agilent cover tubing, which is sometimes covering the HPLC metal tubings (I guess you know what I mean).

After assembling the degasser unit and re-installation to the HPLC unit, all solvent lines were purged with their appropriate solvent mixtures (for us: A1 = 5% AcN/Water 0.01% HCOOH, B1 = 95% AcN/Water 0.01% HCOOH, A2 = 0.02%

NaN3 in water (storage solvent, PBS for HPSEC runs) and B2 = 0.02% NaN3 in water) at 2.5 ml/min until no air was coming out the degasser outlet (approx. 5 min, ~ 10 ml).

The former dirty solvent line A2 was further purged at 0.1 ml/min with 0.02% NaN3 in water over night. Now the pump pressure is stable/no air in the system and no parts coming out the degasser any more. 

Nice work, Marcus! Thanks for writing it up for us all.

Jacek

The main problem you experienced was probably caused by a lack of flushing the HPLC system down at the end of each day. This system is not designed to be left in PBS overnight. The salt content will build up inside the Teflon membranes within the degasser and also in the rest of the HPLC system and result in contamination. It may also contaminate the multi-channel-gradient-valve (MCGV) used with quaternary pumps too, causing problems with other channels. Regular flushing of all of the lines, each day, with DH20 and then some organic added (e.g 20% MeOH) will clean out the system. Also, because you are using a model G1322A degasser with very large 12 ml volume vacuum chambers, you need to take this into account when flushing EACH channel. The low pressure connection tubing adds another 5-8 mls of volume to each channel, so you really need to flush about 60mls of wash solution (3x, because you actually need ~20 mls total for one flush, per channel) through EACH channel at the end of the day (and also when you start the system up each day). This is required to maintain and operate the HPLC system (Part of normal HPLC maintenance/operation). Overnight, gas bleeds back into the Teflon low pressure tubing and these bubbles need to be flushed out before you can start equilibrating the column. Open up the prime purge valve to waste and flush down each channel, one-at-a-time, 100% A-B-C-D. This is something that I teach in all of my intro HPLC classes. Doing this will save you a lot of time and help maintain the machine in a clean, bubble free state.  BTW: if you have an HP/Agilent Quat pump, be sure and set the max pressure to

Hi, Marcus! 

I am experiencing the same problem with possible bacterial contamination in LS system I'm currently working with.  Could you tell me, what was the concentration of the pepsin in 0.1 M HCl solution?

Thank you!

Hi Gabriele,

I used the undiluted solution as from the Link attached (VWR Number 83603.290). Note that this was not the finally solve the problems. The unit got clean only after I pushed liquid through with a very high flow rate (external pump attached - 50 ml/min). This created a force which was sufficient enough to push out the decontaminants.

I strongly recommend you to carefully read Mr. Bill Letter's advises - they are very valuable all the time!

Hope that helps,

Marcus

https://de.vwr.com/store/catalog/product.jsp?catalog_number=83603.290