John W. Dolan
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I remember reading a study on learning in the all-day short-course format. Because teaching liquid chromatography (LC) classes
is a significant part of my work, my attention was captured. The writers claimed that in a 6–8 h class, only three points
would be remembered. One of my LC troubleshooting classes has approximately 200 slides — what does this say about how effective
a short course is at conveying critical knowledge? As the saying goes, I have tried to make lemonade out of these lemons,
and use the "only three things" concept to help reinforce what I think are the key points. So, this month's "LC Troubleshooting"
installment will use these points to form the core of a preventive maintenance program for your LC system.
Degassing
Mobile phase degassing is the single most effective way to avoid problems with an LC system. Liquid chromatographs and air
just weren't meant to be together! LC pumps are very effective at pumping liquids, but introduce an air bubble and in the
best circumstances you will observe a momentary reduction in the flow rate and a drop in the system back pressure. If the
bubble is large enough, the pump will not deliver any solvent, and if the pressure drops below a preset low-pressure limit,
the pump will stop. Some pump designs will pass bubbles fairly well, whereas other designs will fail to operate when a bubble
is present.
Once a bubble passes through the pump, it usually will stay in solution due to the system pressure as it passes through the
column. But on arrival at the detector, the system pressure returns to atmospheric pressure and the bubble might reappear
in the detector flow cell, causing spikes in the chromatogram. This problem can be minimized by the use of a back-pressure
restrictor on the detector outlet to provide sufficient pressure to keep bubbles in solution until they exit the detector.
Of course, care needs to be taken not to exceed the pressure limits of the flow cell, or detector damage can occur. Although noise spikes are the most common symptom of bubbles going through the flow cell, such as with UV detection, some
detectors can be very sensitive to the presence of air. For example, dissolved oxygen has been reported to quench the fluorescence
of some compounds when the fluorescence detector is used (1). In the reductive mode, the electrochemical detector is extremely
sensitive to dissolved oxygen. Care must be taken to eliminate oxygen from the mobile phase and avoid oxygen-permeable tubing
(such as PTFE) in the flow stream.
All of these problems related to dissolved air in the mobile phase can be avoided if proper care is taken to degas the mobile
phase before it is used. For many years, the gold standard for degassing has been helium sparging. This simply involves bubbling
helium through a frit placed in the mobile phase reservoir. Helium sparging is the most effective way to remove dissolved
air from the mobile phase, removing approximately 80% of the oxygen (2). With a well-distributed sparging stream, one volume
of helium will remove almost all the gas that can be displaced from an equal volume of mobile phase (3). This means that 1
L of helium bubbled through 1 L of mobile phase will do the job.
Helium is the only effective way to remove sufficient oxygen from the mobile phase to avoid problems specific to dissolved
oxygen, such as the fluorescence quenching or electrochemical detector problems mentioned earlier. However, if the main objective
is to remove sufficient dissolved air so that bubble formation is not a problem, vacuum degassing is also effective as a degassing
technique. Most of today's LC systems come with an in-line vacuum degasser either as a standard feature or an optional one.
In-line degassing is simple to use, trouble-free, and effective. I give it credit for the huge reduction in bubble-related
complaints that I have heard in the last few years.
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