Degassing your mobile phase is critical to maintaining a stable baseline without fluctuations. Modern electronic vacuum degassing systems are a great convenience. Changing Helium gas cylinders, monitoring gas pressures and levels over time have been replaced in many labs with the sound of small gurgling vacuum pumps. While the convenience gained through the use of these devices can be great, there are some important downsides to this technology. One of them will be mentioned here.
Total Channel Volume: The volume of liquid contained in the tubing and vacuum chamber of EACH vacuum degassing channel can be enormous compared to the tiny volume often found in systems which utilized Helium sparging alone. When the system is turned off, this solvent often sits stagnant over night, allowing air to bleed in, and must be flushed out when the system is re-initialized for use the following day. *If you just switched bottles of mobile phase, then the old mobile phase is still inside the system and needs to be completely flushed out before use or you will have some very strange chromatographic results in the coming weeks! This process takes time and should be performed on every channel in your system, even if you are not going to use them all. How much volume you ask ? Well, let’s look at a few common examples of vacuum degassing units we find at client sites (but this applies to any manufacturer’s system, just look up the spec’s or measure it on your own).
- Agilent/HP Brand Degassing Module, Model G1322A; Each vacuum channel chamber has a volume of about 12 ml. The interconnecting tubing (solvent frit to bottle head, bottle head to degassing unit and degassing unit to pump inlet) can add another 10 mls more. In this example, we recommend that you flow at least 30 mls of the appropriate solvent through the line before use. This will insure that the channel is primed with fresh mobile phase. That step is for EACH channel so it is best to flush them one-at-a-time, in sequential order, to keep track of them.
- Agilent/HP Brand Micro Degassing Module, Model G1379A, G1379B or G4225A: Each vacuum channel chamber has a volume of about 1 ml. The interconnecting tubing (solvent frit to bottle head, bottle head to degassing unit and degassing unit to pump inlet) can add another 10 mls more. In this example, we recommend that you flow at least 20 mls of the appropriate solvent through the line before use. This will insure that the channel is primed with fresh mobile phase. That step is for EACH channel so it is best to flush them one-at-a-time, in sequential order, to keep track of them.
- Waters Alliance 2690, 2695, 2790, 2795, 1525 Systems. Each channel in these modules has a 0.5 ml volume. Because these are integrated degassers (exc for the 1525 stand-alone), the tubing volumes are also low ~ 5 ml each. For this example, we recommend you flush each channel with at least 10 mls of mobile phase before use. Remember to flush them one-at-a-time, in sequential order, for best results.
NOTE: Some early models of HPLC degassing systems had individual chamber volumes of greater than 30 mls each ! When you add the total tubing volume to that you end up with a flush volume of 40 to 50 mls per channel. These systems took a long time to flush out.
To save time performing these “flush” steps, you should take advantage of the highest available flow rate setting of your pump. With the prime purge valve open (so the solvent goes to waste), set the pump flow rate to 5 or 10 ml/min at 100% for the first channel and you will be able to flush out a single channel in two or three minutes. Repeat for each of the other channels and then adjust the flow rate back down to your normal equilibration flow rate before closing the prime purge valve again. Our lab performs this flushing procedure at the start of each day to insure the HPLC columns receive fresh solvent. This procedure should also be performed any time a mobile phase bottle is changed out to a different bottle or liquid is "topped off" too.
Make sure that your degassing module is 100% fully functional. These modules usually wear out after about five years of normal use. Problems with leaks, contamination and failed vacuum pumps are very common. If the unit is repaired early, the cost is often much less than delaying repair so have the system properly diagnosed at the first sign of trouble. Anytime the degasser's performance drops below the manufacturer's specification, your entire HPLC system falls out of compliance. You must repair the degasser module to use it. The degasser is an important component of your entire HPLC system so it must be maintained and serviced just like the other modules. RED or Yellow Error lights (on Agilent/HP models) or "Degasser hardware fault" error (Waters) on these modules always indicates a need for service.
Please keep in mind that running liquid at 10 ml/min during the flush phase in most analytical degassing modules will not result in very efficient degassing as degassing efficiency is a function of flow rate (surface area of liquid exposed over time). You will still need to wait a period of time until the new solvent is fully degassed once again, but at least you will know it is free of any older mobile phase. *Remember, everything you do before you start an analysis contributes to the final result.
- Do you own an Agilent, HP or Waters brand HPLC degasser module which needs parts or to be repaired? Chiralizer Services can repair most HPLC degasser modules, with quick turnaround, for less money than other companies. Please refer to this link for more information: http://www.chiralizer.com/HPLC_Degasser_Repair_Parts_Service.htm