HPLC HINTS & TIPS for Chromatographers
- Tip# 101: INLINE HPLC DEGASSING SYSTEMS:
Modern electronic vacuum degassing modules 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, lets look at two common examples of vacuum degassers we find at client sites (but this applies to any manufacturers system, just look up the specs 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 degasser and degasser to pump inlet) can add another 10 15 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: Each vacuum channel chamber has a volume of about 1 ml. The interconnecting tubing (solvent frit to bottle head, bottle head to degasser and degasser to pump inlet) can add another 10 15 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.
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.
To save time performing this flush step, 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 or if appropriate, to a separate collection container for re-use), set the pump flow rate to 10 ml/min 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 columns receive fresh solvent. This procedure should also be performed any time a mobile phase bottle is changed out to a different bottle or type of mobile phase too.
One last reminder. Please keep in mind that running liquid at 10 ml/min during the flush phase in most degassing systems 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. > Bill Letter 05/29/10.
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