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Comments
The
UV absorbance of dissolved oxygen itself is very small, but it is believed
that dissolved oxygen forms complexes with solvent molecules, and that
these have appreciable absorbance. With mixed solvents, more than one
complex species would be expected. Indeed, with mixed mobile phases more
than one peak is often observed.
You are more likely to encounter artifact peaks when using an efficient degassing system. Oxygen in the air will dissolve in degassed mobile phase very rapidity. The more thorough the degassing, the greater is the driving force to reabsorb oxygen. If peaks appear when they have previously been absent, check to see if a procedure has changed that might have changed the oxygen concentration in either the mobile phase or the sample. Oxygen can enter the mobile phase by diffusing upstream against the outflowing helium in the reservoir vent tubes and by diffusing through Teflon® reservoir-to-pump transfer tubes.
Oxygen Peak Artifacts
Artifact peaks are sometimes produced when the sample (or blank) contains oxygen in a concentration that differs from the oxygen concentration in the mobile phase that is flowing at the time of injection. This difference is common when the mobile phase is completely degassed, such as when continuously sparged with helium. The mobile phase arriving at the injector will contain essentially no oxygen, but the solvent in which the sample is dissolved will contain oxygen unless it has been degassed immediately prior to injection.Such artifacts are particularly noticeable when a UV detector is operated at high sensitivity and at a low wavelength. They also occur with electrochemical detectors, where the peaks can be positive or negative.
The following procedure will show if oxygen is the problem. First remove a 1 mL aliquot of mobile phase from the chromatograph's reservoir. Immediately inject a blank using some of this solvent. Observe the peaks. They may be very small or nonexistent. Now agitate the remaining aliquot for at least two minutes to equilibrate it with air. Inject another blank using this solvent. Observe the peaks. If the peaks of the second injection are larger, and correspond to the retention times of the artifacts, it indicates that the problem is differences in oxygen concentration.
You can further confirm this by making blank injections of mobile phase that contain differing amounts of oxygen. For example, compare injections of mobile phase that has been sparged (in the vial prior to taking it into the syringe) with helium, nitrogen, air, or pure oxygen. The relative amount of oxygen in these three gases is 0.0, 0.2, and 1.0, so the peak heights in the three cases should have this ratio.
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