The introduction of electronic vacuum degassing / degasser modules to the liquid chromatography industry a few decades ago has introduced several new problems which were unknown years ago when we sparged our mobile phase solutions with high-purity helium gas. One of these problems relates to how the electronic vacuum degassing modules themselves can contribute to contaminating your HPLC or LC-MS system.
Before
using an HPLC vacuum degasser module, please review all of the information
and advice supplied by the manufacturer of your specific degassing module. The
composition of the internal degassing tubing has changed a great deal over the
past decades resulting in increased degassing efficiency, but also
changes in mobile phase chemical compatibility. Some popular solvents may be
incompatible with some models in your lab. Make sure you know exactly
which types of vacuum chambers and plastics are used in your specific
instrument(s). Degassing modules must be operated, cleaned and
maintained the same as your other important instruments. When they are
not operating properly and/or are contaminated, they should be serviced as
soon as possible or risk further contamination and damage to your system
($$$).
- Link to a pdf document with pictures showing common contamination and damage observed while inspecting HPLC Vacuum degasser modules for our clients. Have you had your HPLC vacuum degasser(s) professionally evaluated for damage? [ https://www.chiralizer.com/examples_of_degasser_contamination.pdf ].
In a previous post ["Inline HPLC Degassing Modules"] we discussed the convenience that these devices have brought to our laboratories, but also the extra training requirements (such as cleaning and flushing the vacuum channels every day and routine servicing every 2 to 5 years) 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 internal wettable surfaces of each degassing chamber contain plastics (examples of plastic used: Teflon, Teflon AF, Tefzel and/or Peek are the most common types of used). To effectively remove gas from the mobile phase, the liquid must pass through plastic tubing (or across membranes) placed in a vacuum, for a period of time which is long enough to allow a portion of the dissolved gas in the mobile phase to diffuse through the degassing tubing/membrane and out the exhaust port of the degassing chamber. 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 type of fluoropolymer tubing (Variants of Teflon or Teflon AF) being used as they have broad chemical compatibility plus can be formed with controlled pore sizes for the effective removal of gas, not liquid, through the tubing walls. However, there are exceptions to this and the plastic(s) used may NOT be chemically compatible with all liquids used in chromatography applications. Depending on the plastic degassing tubing used, the tubing may swell, fail or even dissolve into the mobile phase solution! Be sure and check
the chemical compatibility chart offered by the degassing module
manufacturer for compatibility with your mobile phase and ALL additives
used before using the instrument. Some examples of incompatible
chemicals on the lists of many instrument vendors include: THF, Chloroform,
DCM, strong acids or bases, Hexanes and Sodium Azide. Use
of incompatible solvents or additives may result in complete failure of
the degasser module plus contamination of the entire instrument flow
path. We have seen many degasser systems which were used with (or stored in, w/o proper flushing) strong acids show corrosion of the metal parts inside the chambers (SS fittings and connectors) forming piles of rust and salts which were carried through the vacuum system resulting in damage to the system and flow path contamination. *Please do not risk it. Be aware of which chemicals may pose a
risk with your system. For example: The use of many fluorinated solvents may dissolve most types of tubing when Teflon AF is used for degassing.
- Note: We have seen an increase in the use of various perfluorinated solvents, esp with LC/MS systems. This has resulted in severe degasser damage plus MS source contamination (e.g. HFIP and Ethoxynonafluorobutane). Most perfluorinated solvents are not compatible with vacuum chambers which contain Teflon AF. They may dissolve the degassing tubing, resulting in the destruction of the degasser chambers and contamination of the vacuum system and mobile phase (IOW: the complete HPLC system flow path). 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 signal). These ion-pairing 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) alternated with a wash containing some base (ammonium) often helps in this regard. Wash cycles of over 12 hours are often needed to remove these compounds and see improvement (It may take much longer...). In some cases we must replace some or all of the internal parts of the degassing module to eliminate the contamination. Always remove any HPLC column from the flow path (to avoid re-contaminating again) and replace with a new one, once the contamination has been removed. It is for this reason that we should avoid the use of strong ion-pairing reagents in any LC/MS system, as they often contribute to very high background signals and long term contamination. *Helium sparging should be considered for such applications.
Reversed phase HPLC applications which use highly aqueous mobile phases may under some circumstances result in high rates of pervaporation of the water vapor into the degasser module resulting in condensation of the water into the vacuum system (Unlike the older Teflon material used, the newer Teflon AF formula is more permeable to water vapor). Once liquid enters the vacuum pump, severe damage has already occurred and failure of the vacuum system soon follows. *If you ever notice liquid exiting through the vac pump's exhaust port, turn off the HPLC system and have the degasser module professionally serviced.
Another common problem seen when aqueous solutions are used in an in-line vacuum degasser are that of algae and bacterial growth. Most often observed in systems left unused for a period of time or which are not periodically flushed out with organic solvents. Growth inside the low pressure tubing and even inside the vacuum chambers has been known to contaminate the entire flow path of an HPLC system. Replacement of the tubing and internal chambers usually resolves the problem.
Buffers / Additives: Just as with the rest of your HPLC system, any buffer salts, acid, bases or additives which are left in the system (even overnight) can damage it. This is true of the vacuum degasser module too. Please be sure to flush all of the vacuum degassing chambers of any salts or buffers when not in use.
For normal phase applications, high concentrations of n-Hexane may cause contamination or damage to a degasser attached to an HPLC or LC/MS system. Some types of degassers are not compatible with Hexane. The ultra high evaporation rate of hexane coupled to the advanced materials found in the degassing tubing or membranes may result in the hexane condensing on the outside of the internal degassing tubing of the degasser and then aspirated into the vacuum system (causing damage). The contaminants are then transported back through the tubing walls into the solvent stream (your mobile phase).
If your HPLC's vacuum degasser fails to achieve vacuum, has liquid exiting the vacuum pump exhaust port (or exhaust tubing) or shows an error (e.g. Leak Error, High RPM, makes loud noise, a Yellow or Red light on an HP/Agilent system or "Degasser Hardware Fault" / "Degasser High Leak Rate" messages often seen on Waters brand systems), then your entire HPLC system may be out-of-compliance - because the degasser is broken. Have the vacuum degasser professionally cleaned and repaired so you can put the system back online. Do not assume that only the vacuum pump has failed, as replacement of the pump alone often results in failure of the replacement pump soon after (due to contamination and other problems incorrectly diagnosed). The true cause of the failure must be correctly diagnosed and repaired first, and this is something best left to professionals.
Our professional HPLC degasser repair shop receives many types of degasser modules with leaking or ruptured vacuum chambers. These directly 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 and vapors 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, but MS source too (costing a great deal of money to decontaminate). Please, at the first sign of trouble, have the degasser professionally diagnosed, cleaned and repaired. For more information on having your degasser professionally diagnosed and repaired with fast turnaround at a fraction of the price charged by most instrument vendors, please refer to this link: "HPLC Degasser Repair Service" [ http://www.chiralizer.com/hplc-degasser-repair.html ]
