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Saturday, August 1, 2015

An Often Ignored HPLC & LC/MS Contamination Source. Did you check your Vacuum Degasser?

The introduction of electronic vacuum degassing / degasser modules to the liquid chromatography industry a few decades ago has introduced several new problems which were not seen years ago when we sparged our mobile phase solutions with helium gas. One of these problems relates to how vacuum degassing modules can contribute to contaminating your HPLC or LC-MS system.

In a previous post ["Inline HPLC Degassing Modules"] I discussed the convenience that these devices have brought to our laboratories, but also the extra training requirements (such as cleaning and flushing the channels every day) which must be undertaken to use them successfully. When the operational guidelines for the use of these products are ignored, these devices can contribute to the contamination of your HPLC and / or LC-MS system. The wettable internal surfaces of the degassing module contain one or more types of plastic(s) which come in contact with the mobile phase (AF Teflon, Teflon and/or Peek are the most common "wettable materials" found). To effectively remove gas from the mobile phase, the liquid must move through plastic tubing or pass by membranes, placed under vacuum, for a period of time which is long enough to allow the gas to diffuse through the material and out the exhaust port of the degassing module. The degassing tubing (most use tubing) should have the maximum chemical compatibility possible while allowing it to also be porous enough for the gas alone to diffuse through the walls of the tubing under vacuum. These requirements usually result in some form of fluoropolymer tubing (AF Teflon or Teflon) being used as they have broad chemical compatibility and can be formed with controlled pore sizes for the effective removal of gas, not liquid, through the tubing walls. However, their are exceptions to this and the plastic(s) used may NOT be chemically compatible with all liquids used in chromatography. Depending on the plastic used, the tubing may swell, fail or even dissolve into the mobile phase solution! In many cases, the use of fluorinated solvents will dissolve these materials (esp. Teflon AF). We sometimes see a white residue leaking out of the degassing outlet ports which is initially thought to be buffer salts (very common from a lack of flushing), but sometimes it turns out to be dissolved tubing! Be sure and check the chemical compatibility chart offered by the degassing module manufacturer for compatibility with your mobile phase and ALL additives before using the instrument. Some examples of incompatible chemicals on the lists of many vendors units include: THF, strong acids or bases, Hexanes, Sodium Azide and especially most fluorinated solvents which can dissolve many of the plastic parts inside. *Be aware of which chemicals may pose a risk with your system. Contaminated vacuum chambers quickly result in contaminated tubing and vacuum pump failure.

Over the past few years we have seen an increase in the use of various perfluorinated solvents, esp with LC/MS systems and this has resulted in some serious degasser damage and source contamination (e.g. HFIP). Additionally, we commonly see ion-pairing reagents such as TFA and TBA "sticking" to the plastic used in these modules causing a leaching of material over long periods of time (again, most obvious on an MS system where you can "detect" it in the background). These agents must be thoroughly flushed out of the flow path to reduce contaminating the entire system over time. *A strong wash solution with a little acid (formic) often helps in this regard. Wash cycles of over 12 hours are often needed to see improvement. In some cases we must replace some or all of the internal parts of the degassing module to eliminate the contamination. 

For normal phase applications, high concentrations of n-Hexane can also cause contamination of an HPLC or LC/MS system. The damage is often sourced to the degasser (which are often not compatible with Hexane) where the ultra high evaporation rate of hexane coupled to the advanced materials found in the degassing tubing results in the hexane condensing on the outside of the tubing of the degasser and becoming contaminated with the exhaust and plastics found there. These contaminants are then transported back through the tubing walls into the solvent stream.

If your HPLC's degasser has an error light turned on (i.e. Yellow or Red light or "Degasser Hardware Fault" error message), then your entire HPLC system may now be out-of-compliance because the degasser is broken. Have the degasser professionally repaired so you can put the system back online. Maintain these devices, following the manufacturer's guidelines in their use and operation and you should be able to run them for about five years before requiring repair service.

Our HPLC degasser repair shop also receives degasser modules with leaking or ruptured vacuum chambers. These too contribute to mobile phase contamination as any seal failure in these normally "dry" systems results in liquid contaminating the vacuum system which in-turn sends contaminated liquid back into the HPLC mobile phase stream. *Note: If you are using your HPLC degasser with Mass Spec detector, then the resulting mobile phase contamination may contaminate not just your column and MS source (costing a great deal of money to decontaminate). Please, at the first sign of trouble, get that degasser cleaned and repaired.

Be sure and review all of the information and advice supplied by the manufacturer of your degassing module before use or whenever you use any new solvents or additives. The composition of the degassing tubing has changed a great deal over the past decades resulting in increased degassing efficiency, but also changes in chemical compatibility. Certain popula solvents may be incompatible with most models in your lab. Make sure you know exactly what types of vacuum chambers and plastics are used in your specific instrument(s) and proceed with caution when using these systems. Degassing modules must be operated, cleaned and maintained the same as your other important instruments. When they are not operating properly or are contaminated, they should be serviced as soon as possible or risk further contamination and damage to your system ($$$). 


  1. Very good article and a common challenge, especially when systems are started up again after a longer period of not using the unit. Iso-propanol (HPLC grade) has proven to be a good cleaning solvent but also storage solvent for vacuum degassers but also the entire system. For normal phase and very small column flows (< 5 uL/min) I would bypass the vaccuum degasser.

  2. Big help! We had white crystals forming on the outlets from our degassing unit going into the pump and did not know what it was. No one every told us we needed to flush the buffers through it. Now we flush the system every day with water as you suggest and it works so much better. Before, we had baseline going all over the place. Some said it was the check valve, but valve was fine. We bypassed the degass and it started working again. So now we flush it with the right liquids and never leave buffers in the system. Works great now for two weeks! Thanks for these great posts.