Why should you be concerned about mobile-phase degassing — it's all done automatically, isn't it?
Degassing of the mobile phase in liquid chromatography (LC) applications is a topic that is seldom talked about today. However,
this was not the case in the past. For the first 30 years or so of LC use, problems related to mobile-phase outgassing were
some of the most common problems encountered in routine use. This was true in user surveys conducted by LCGC as well as by my informal polls based on readers' questions and direct interactions with users. Over the past 15–20 years,
on-line degassers have moved from a novelty device to a standard part of most LC systems. As a result, I suspect that many
users have never encountered problems related to degassing. I dropped an in-depth discussion of degassing from my popular
LC troubleshooting class, but in a recent class the topic came up again. Although in-line degassing helps us avoid most solvent
out-gassing problems, it does not solve all problems related to dissolved air in the mobile phase. In this month's installment
of "LC Troubleshooting" I would like to review what degassing is all about. What is it? Why do we need it? How is it accomplished?
Are there times when we should be especially watchful for problems related to it?
Why Degas the Mobile Phase?
When solvents are in contact with the atmosphere, air gradually dissolves into the solvent. Air, of course, is primarily nitrogen
and oxygen. In reversed-phase LC, the most common solvents are water or buffer as the A-solvent and acetonitrile or methanol
as the B-solvent. When the aqueous and organic solvents are mixed, they each contribute to the total dissolved air content
of the mixture. This is illustrated by the dashed line in Figure 1 for the solubility of oxygen in mixtures of water and ethanol
(which behaves in a similar manner for oxygen and nitrogen with methanol and acetonitrile for the present discussion). Whether
we are making isocratic (constant %B) mixtures or gradients, the amount of gas in solution is in proportion to the respective
solvent volumes. The problem with this situation is that the solubility of air in the mixture is less than that of individual
components. This is shown as the solid curve in Figure 1. When such a situation exists, the solution is supersaturated with
air, generating an unstable condition in which air will bubble out, or outgas, from the solution.
Figure 1: Solubility of oxygen in ethanol (dashed line); saturation concentration of oxygen in the mixture (solid line).
See text for details. Adapted from reference 1.
If mobile-phase outgassing occurs within the LC system, the most common problem areas are the pumps and detector. At the extreme,
air in the pump will cause the pump to stop delivering mobile phase to the column. If only an occasional bubble is present,
the flow rate will be erratic, causing retention time problems. Air in an optical detector, such as an ultraviolet (UV), fluorescence,
or refractive index detector, will scatter light passing through the flow cell, causing noise spikes in the chromatogram or
an off-scale signal. These problems can be eliminated if the air is removed from the mobile phase.
The data of Figure 1 suggest that to avoid mobile-phase outgassing we do not have to remove all the dissolved gas from solution,
but only reduce the total amount of dissolved gas so that a plot for the resulting mixture would result in a line falling
below the solid line in Figure 1 (1). If half of the dissolved gas is removed, we should be safe. One simple way to do this
is to use a vacuum to degas the solvents. This can be done easily by placing the solvent in a vacuum flask and pulling a partial
vacuum with a water aspirator or mechanical pump of similar capacity. To help facilitate the process, a stir bar or a few
(clean!) boiling stones can be added to the flask. Although I don't have quantitative data on this, many workers find that
sonicating the solution while vacuum degassing seems to be more effective than vacuum alone. In any event, a few minutes of
vacuum degassing will remove about 60–70% of the dissolved gas (2). Sonication alone will only remove 20–25% of the gas (2),
which is insufficient to avoid outgassing with most LC systems. For some LC systems, the amount of vacuum applied while filtering
the mobile phase may remove enough gas to avoid problems. The most effective way to degas the solvents is to bubble helium
through the mobile phase by sparging for a few minutes, which removes approximately 80% of the dissolved air (2). It seems
contradictory to use a gas to degas a solution, but the solubility characteristics of helium in the mobile phase are such
that outgassing is not a problem. Helium sparging was widely used for degassing, and although it is used less today because
of the ease of in-line vacuum degassing and decreased availability of helium, it is still the most effective degassing technique.
Until the late 1970s, all LC systems either were run with premixed mobile phases in the isocratic mode or used high-pressure
mixing to generate a gradient by mixing the mobile-phase components after the pumps. With these systems, the premixed mobile
phase was degassed before use or the individual solvents were degassed for gradient applications. On-line mixing of isocratic
mobile phases was also possible if the solvents were degassed first. The advantage of high-pressure mixing is that the pumps
only pump degassed solvents or mobile-phase mixtures, and because the solvents were mixed under pressure, any bubbles that
might otherwise form at atmospheric pressure would stay in solution because of the elevated pressure of the system. Often,
care had to be taken to provide a small back pressure (for example, 50 psi) on the outlet of the detector to keep the mobile
phase from outgassing in the detector cell.
In the late 1970s, Spectra-Physics (now a part of Thermo Fisher Scientific) introduced an LC system that incorporated low-pressure
mixing. Solvents were blended in a proportioning manifold before they reached the pump, a technique that is used in low-pressure
mixing systems offered by most LC manufacturers today. When low-pressure mixing is used, the solvents are mixed at atmospheric
pressure (or sometimes a bit below atmospheric pressure), so outgassing of the mobile phase is a huge problem. The Spectra-Physics
system included a built-in helium sparging system to degas the solvents before mixing, so outgassing was avoided. Although
Spectra-Physics had a patent on helium sparging (3), which discouraged other manufacturers from offering the same technology,
it was hard to prevent individual users from constructing their own helium sparging apparatus for personal use, so the technique