HPLC degasser issue

[FreshLC]

Hey everybody,

first time poster here. So our lab is stuck in quite the predicament. There is definitely something wrong with our Agilent 1260 system, specifically the degasser. The reason why i'm saying that is today I disconnected the inlet/outlet in/from the degasser and the liquid which came out and absorbed on my paper left brown/reddish stains. If I keep the system running on 50/50 methanol/acetonitrile the pressure constantly rises until the autosampler in-line filter completely clogs up with the very same brown/reddish material. Pump is a G7104C quat pump from Agilent, the degasser is an integrated one, which makes it even more difficult for me to pick it apart.

My only ideas would be some sort of bacterial contamination or that some plastic material inside the degasser has desolved and is causing me all the trouble. I doubt the second option though, as I checked what the manual says about what polymers the degasser is made of and no solvent would be able to cause damage to such materials (Teflon AF, FEP, PFA, ETFE). Only thing I could consider harmful would be fluorinated ion pairing reagents as HFBA/TFA that were ran on that system (up to 0,3 and 0,1% respectfully) although that really can't do such amount of damage. Colleague of mine also ran diethylamine/triethylamine/ammonia in his experiments as mobile phase additives but I doubt once more that could have done this much harm.

Apart from that the pump is fully functional, no issues whatsoever concerning leaks and further more. Everything that was ran on that system in the past year was water soluble. Buffers are out of the question, last time a buffer was used there was November.

I'd be more than happy if anyone could point me in the right direction, any help is highly appreciated.

[Consumer Products Guy]

You're a lab, you should be able to do a spot test for iron/rust, even a spot test.

[Multidimensional]

The red/brown debris you are seeing coming out of the degasser outlets is from the degasser vacuum pump disintegrating. This is due to lack of preventative maintenance and/or abuse. That debris has now contaminated one or more of the degasser vacuum chambers too (so your mobile phase and column are now contaminated). Cite: "An Often Ignored HPLC & LC/MS Contamination Source. Did you check your Vacuum Degasser?"; https://hplctips.blogspot.com/2015/08/an-often-ignored-hplc-lcms.html

You are correct that fluorinated solvents will destroy the degasser chambers. So will use with hexane, DCM, chloroform, THF, sodium azide and many other solvents and compounds. They are very delicate and everything you run through the mobile phase goes into the degasser (via condensation) causing cross-contamination when they are damaged. These newer style degasser modules are very fragile compared with the older styles. Use of the newer style degassers requires more training and regular servicing and cleaning to keep them running.

*Make sure everyone in your lab knows to avoid those liquids and compounds when using these.

**So many people ignore these critical devices, esp when they are installed / integrated into the pump module (you cannot see them). HPLC vacuum degassers require professional PM service at least every 5 years (or sooner, anytime a problem is observed). The 'fix' involves having the degasser module professionally serviced. This involves removing the degasser module from your pump (it is its own self-contained plastic/SS box and can be taken out to send for service). The "box" can be serviced and then returned to you for installation (instructions are in the manual). DO not try and service it yourself. Proper service requires specialized equipment and experience to test and evaluate each part (sensor, chambers, pump, tubing, electrical).

Recommendation: Now Agilent will be happy to charge you $4 to 5k plus to come in and swap the module out, but if you are interested in saving thousands of dollars and having the degasser professionally tested and serviced, then contact Chiralizer Services, LLC (USA) for more information. They can find out what is actually wrong with it and then decontaminate and service it for a small price compared to what the manufacturer charges, plus has fast turnaround times (~ 2 days) too. The average repair is ~ $1K.

More info: http://www.chiralizer.com/hplc-degasser-repair.html

[vmu]

sodium azide

What about 0.02-0.05% sodium azide in water used as a storage eluent for many SEC columns?

[FreshLC]

The red/brown debris you are seeing coming out of the degasser outlets is from the degasser vacuum pump disintegrating. This is due to lack of preventative maintenance and/or abuse. That debris has now contaminated one or more of the degasser vacuum chambers too (so your mobile phase and column are now contaminated).

You are correct that fluorinated solvents will destroy the degasser chambers. So will use with hexane, DCM, chloroform, THF, sodium azide and many other solvents and compounds. They are very delicate and everything you run through the mobile phase goes into the degasser (via condensation) causing cross-contamination when they are damaged. These newer style degasser modules are very fragile compared with the older styles. Use of the newer style degassers requires more training and regular servicing and cleaning to keep them running.

*Make sure everyone in your lab knows to avoid those liquids and compounds when using these.

**So many people ignore these critical devices, esp when they are installed / integrated into the pump module (you cannot see them). HPLC vacuum degassers require professional PM service at least every 5 years (or sooner, anytime a problem is observed). The 'fix' involves having the degasser module professionally serviced. This involves removing the degasser module from your pump (it is its own self-contained plastic/SS box and can be taken out to send for service). The "box" can be serviced and then returned to you for installation (instructions are in the manual). DO not try and service it yourself. Proper service requires specialized equipment and experience to test and evaluate each part (sensor, chambers, pump, tubing, electrical).

Recommendation: Now Agilent will be happy to charge you $4 to 5k plus to come in and swap the module out, but if you are interested in saving thousands of dollars and having the degasser professionally tested and serviced, then contact Chiralizer Services, LLC (USA) for more information. They can find out what is actually wrong with it and then decontaminate and service it for a small price compared to what the manufacturer charges, plus has fast turnaround times (~ 2 days) too. The average repair is ~ $1K.

More info: http://www.chiralizer.com/hplc-degasser-repair.html

Are you certain it is the degasser chamber? According to Agilent it should be compatible with Hexane, DCM etc. There was even an Agilent technician once here who recommended washing the system in case it gets clogged up with 50/25/15/10 IPA/MeCN/Hexane/DCM. I checked for rust, test came out negative so thats out of the question. None of the red brownish stain is visible anymore too, if it were the degasser pump, wouldnt I be seeing that non-stop? I checked all the system frits for any of the same residues and they were clean compared to a few days ago. I also had the residue coming out of only one channel so I suppose it might have indeed been some sort of bacterial contamination.

[Multidimensional]

Sodium azide is listed on the manufacture's chemical compatibility list as "INCOMPATIBLE" for the vacuum chambers used in the pump you have. PLEASE note that AGILENT Technologies is NOT the manufacturer of the degasser used in your pump.

[Multidimensional]

"Are you certain it is the degasser chamber? According to Agilent it should be compatible with Hexane, DCM etc."

PS I never said it was 'rust' (that was someone else).

We can not be certain it is one of the degasser chambers until the degasser has been fully tested and evaluated to find out what is actually wrong with it. If it is more than 5 years old, the vac pump is probably damaged and contaminated. My comments are based on what is likely, but without examining it, we can not know for certain. Disclaimer: Please note that I work for an HPLC & LC-MS instrument company who also specializes in the servicing of all brands of HPLC vacuum degassers (our company has been servicing vacuum degassers for all of the major instrument manufacturers for ~ 25 years and we have serviced thousands of these systems so are aware of what problems they have and how people use them). The actual chemical compatibility for the vacuum chambers, tubing and vacuum pump and different for what you are reading. You are reading which wetted parts are used and you are not taking into account that the mobile phase vapors are also in the vacuum system so in fact may damage the vacuum system too. The chambers you have are made from PEEK, so are not compatible with many solvents (THF, chloroform....). The standard chambers are not compatible with solvents like Hexane either (per the manufacturer and we have seen hundreds of these systems destroyed by use with Hexane/Heptane over the years). Agilent knows this and is one reason why they still sell the very rugged model G1322A degasser (which is compatible with Hexane and Heptane and THF...). Many of their tech have no training for the degasser and are providing advice for the HPLC system, w/o the degasser. Such comments as; "There was even an Agilent technician once here who recommended washing the system in case it gets clogged up with 50/25/15/10 IPA/MeCN/Hexane/DCM" are NOT applicable to the degasser (NEVER Flush any vacuum degasser with such a mixture unless you are certain it is compatible with it). Your degasser is not compatible with some of those liquids. That flushing mixture was a generic mix used to wash the flow path of an HPLC system (w/o any degasser plumbed inline).

Agilent does not "repair" these, they swap them out with replacement modules. If money does not matter, simply have Agilent replace the degasser module. Also before using the replacement degasser: clean the solvent bottles, install new solvent pickups, then replace all of the Teflon tubing going from the bottles to the degasser and from the degasser to the pump head(s) too (to remove any existing contamination). Next, flush the entire flow path of the system.

If you would like to save money, then contact Chiralizer Services regarding their special bench repair service for HPLC vacuum degasser modules. They have all the parts in-stock and know these products better than anyone.

[Multidimensional]

You also wrote: "system running on 50/50 methanol/acetonitrile ". Why would you run your system in this odd mixture of pure organic solvents? If you desire to flush or store the HPLC pump module, please use a mixture of ~ 30% RO Water: 70% MeOH for storage or general flushing. The water is needed to dissolve any deposits inside. If you only run pure organic, you will allow for build-up and fouling-plugging of the valves, filters and ports inside this expensive pump. For routine flushing, please use a solvent mixture that is appropriate for YOUR samples (that will dissolve them 100%!) to remove any residues from the flow path. There is no single 'universal' flush solvent (and do not let anyone tell you there is). The best wash solution must be determined based on how YOU use the instrument.

Use of any fluorinated ion-pairing (or otherwise) compounds with vacuum degasser Teflon AF membranes is not advised by DuPont or any of the distributors/licensed manufacturer's/vendors. Those materials may dissolve or damage the degassing membranes resulting in contamination of the HPLC system flow path or damage to the chamber membrane (requiring replacement). We have seen cases where it resulting in high condensation rates through the membranes destroying the vacuum system and washing all the debris back into the HPLC flow path. If you wish to use those types of compounds, then you need to use another type of degassing method (e.g. Helium sparging) or use the Agilent model G1322A degasser which does not use Teflon AF membranes and is fully compatible with fluorinated solvents/additives.

[Consumer Products Guy]

Why would you run your system in this odd mixture of pure organic solvents?

We were R&D and manufacturing support. Some HPLC systems ran cGMP stability tests. We had most of our HPLC units having 1 reservoir of methanol, 1 of ACN, 1 of an acidic buffer, and one miscellaneous. All Agilent, and we had very few mechanical issues or check valve issues due to pure organic being used. Just my experience, about 8 HPLC systems, 1050, 1100, and more modern.

[Multidimensional]

That response does not address the oddness of running an HPLC method using 50% MeOH plus 50%ACN as a mobile phase. This has nothing to do with which solvents bottles are attached to your instrument. it has everything to do with the fact that the OP is brand new to HPLC use. PS : I would not expect any check valve issues using such liquids in any combinations on an Agilent HPLC system. The OP was running this odd mix and wondering why they were clogging up their system (because that solution is not an ideal "flushing" solution or proper way to troubleshoot a clog).

[vmu]

Sodium azide is listed on the manufacture's chemical compatibility list as "INCOMPATIBLE" for the vacuum chambers used in the pump you have. PLEASE note that AGILENT Technologies is NOT the manufacturer of the degasser used in your pump.

Agilent 1260 Infinity High Performance Degasser
https://www.agilent.com/cs/library/user ... USR_EN.pdf
Materials in contact with solvent: TFE/PDD Copolymer, FEP, PEEK.
Fittings: ETFE.
"If the application permits add 0.0001 – 0.001 M sodium azide to the solvent".
Also there are no restrictions on aliphatic hydrocarbons such as hexane for this degasser.

Update: about the pump from the first post.
Agilent 1260 Infinity II Flexible Pump (G7104C).
https://www.agilent.com/cs/library/user ... 4CUser.pdf
"How to Prevent and/or Reduce the Algae Problem.
If possible add a few mg/L sodium azide or a few percent organic solvent to the aqueous mobile phase".
Materials in contact with solvent: TFE/PDD copolymer, FEP, PEEK, PPS, stainless steel, polyimide, ceramic, HMWPE.
The only restriction on hexane is described in the following part:
"Polyethylene (PE). ...UHMW (ultra-high molecular weight)-PE/PTFE blends for yellow piston and wash seals, ... for normal phase applications in 1260 Infinity pumps. ... It has limited stability with aliphatic, aromatic and halogenated hydrocarbons... For normal phase applications, the maximum pressure should be limited to 200 bar".
It's difficult to imagine NPLC without aliphatic hydrocarbons.

[Multidimensional]

VMU wrote: "

Agilent 1260 Infinity High Performance Degasser
https://www.agilent.com/cs/library/user ... USR_EN.pdf
Materials in contact with solvent: TFE/PDD Copolymer, FEP, PEEK.
Fittings: ETFE.
"If the application permits add 0.0001 – 0.001 M sodium azide to the solvent

".

None of the above information applies to this issue.
VMU, you have replied with information that is not applicable to the OP's HPLC system or degasser. The information you quoted and reference are for the standalone, Agilent model G4225A degasser (looks similar to the model G1322A, but with different chambers inside (PEEK), new part number). The OP has a 7104C integrated degasser module. As I already noted, the standalone model G1322A degasser is completely different and IS compatible with hexane, sodium azide etc. This is not the integrated degasser that the OP has (I refer to the G1322A), but the one Agilent (and I also) recommend when you plan on using any of the many incompatible solvents noted. *The "other" degasser models have very fragile vacuum chambers, not suitable for some applications.

[vmu]

VMU, you have replied with information that is not applicable to the OP's HPLC system or degasser.

I've added info about G7104C to my previous post.

[Multidimensional]

Most of those notes in the manual apply to the "pump", not the integrated degasser which has Teflon AF and PEEK inside. Agilent assumes the degasser is by-passed when discussing it in their manuals, esp for flushing or troubleshooting (much of the info in manuals was copied from previous pump versions which used a separate, stand-alone degasser, so it did not apply). I have provided the info that pertains to the integrated vacuum degasser module that the OP was interested in (which is not made by them).

[vmu]

It's strange to me that Teflon AF and PEEK cannot tolerate 500 ppm NaN3 or pure hexane.