PerkinElmer 200 UV/VIS Series, User Manual

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Maintenance Troubleshooting Detector Problems
7 - 16
Mobile Phase Degassing
Degassing refers to the removal of dissolved gas from a liquid by mechanical means. Three
methods are commonly used.
• Vacuum degassing
• Sonication
• Helium Degassing
In vacuum degassing, the mobile phase is placed in a vacuum vessel and vacuum is
applied. As the pressure above the liquid drops, dissolved gases come out of solution and are
drawn off. Care should be taken to ensure that the vacuum is not sufficient to bring the sol-
vent, at ambient temperature, to its boiling point. This may cause preferential loss of lower
boiling point components of the solvent mixture with subsequent chromatographic changes.
It is also advisable to combine vacuum degassing with filtration, using a 0.2 - 0.4
µ
m filter,
since this protects the chromatographic hardware (pump, injector, detector, etc.) and column
from plugging by particulate matter.
Sonication may also be used to accomplish degassing. Place the vessel containing the
mixed mobile phase into a sonicator containing about 1/2-inch of water (or more, depending
upon the size of the mobile phase vessel) for 5 to 10 minutes Sonication will eliminate most
dissolved gas from the solvent and may be combined with vacuum for even faster degassing.
Sonication will only last 4 to 5 hours until atmospheric gases reenter the solvent. Solvents
should therefore be sonicated each day.
Once gases in a solvent are removed, steps must be taken to prevent reabsorption from the
laboratory atmosphere. Helium Degassing accomplishes this by sparging (continuously
bubbling) a pure, relatively inert gas such as helium through the mobile phase after degas-
sing and during runs. Even though some of the sparging gas may dissolve in the mobile
phase, levels will be low and constant, eliminating drift and instability.
HPLC mobile phases should be degassed after
mixing! The mechanical action of thoroughly
mixing mobile phase components may reintro-
duce atmospheric gases back into solution.