1. Appearance of Peaks Likely Caused by Dissolved Air (Oxygen)
Figure 1 shows the results from injecting a methanol/water mobile phase mixture directly as the sample, while it is being degassed by online degassing. The concentration of dissolved oxygen was changed and the results compared. If the sample (mobile phase) is injected as-is, in other words still saturated with air, one peak appears (see Figure 1a, where a peak of about 10 mAU occurs from a 10 µL injection). This peak almost disappears if the sample (mobile phase) is injected after purging with helium (concentration of dissolved oxygen is almost zero) (Figure 1b). If purged with oxygen (with a dissolved oxygen concentration about 5 times higher than when saturated with air), the peak size increases. (Figure 1c)
If the mobile phase is not degassed by online degassing, the peak hardly appears at all, even if the sample (mobile phase) is injected saturated with air (Figure 2a). If the sample is purged with helium, a negative peak is observed (Figure 2b).
Based on these results, the peak appears due to a difference in the amount of dissolved oxygen in the mobile phase and sample solution.
2. Peak Size
What about other solvents? Figure 4 shows the difference spectra for various solvents, calculated by subtracting the degassed solvent spectra from air-saturated solvent spectra. For each solvent, absorption increases due to dissolved air, but the dissolved air has a smaller effect on water and acetonitrile and a larger effect on hexane, methanol, and THF.
The amount of change in absorption does not match the level of dissolved oxygen in each solvent. For example, hexane has a much higher oxygen solubility than methanol2), but the change in absorption is small. Therefore, the reason for the absorption is not due to absorption by oxygen alone. Rather, presumably it is a result of an interaction between the oxygen and solvent2).
3. Peak Elution Position
Note that the peak size decreases as the methanol ratio is reduced.
4. How to Check Peaks Assumed to Be Caused by Dissolved Air
- When the sample solvent and injection volume are the same, the peak appears at approximately the same position and size. To saturate the sample solution with air, use a stirrer (or mix by shaking) or leave it in a semi-open system. If the solution is diluted to inject twice the volume, the area is about 2 times as large.
- The elution time of a peak that appears when mobile phase (saturated with air) is injected, matches the elution time of the suspicious peak. In this case, if the mobile phase is degassed (purged with helium for about 10 seconds) and then injected, the peak becomes smaller.
- When mobile phase is delivered without using a degassing unit and the sample solvent is injected, the peak becomes smaller. If air-saturated mobile phase is injected, the peak always becomes smaller than in case 2. If the composition of the sample solvent is significantly different from the mobile phase, the peak does not necessarily become smaller.
5. Corrective Measures
- Change the mobile phase from a methanol-based to an acetonitrile-based (HPLC grade) mobile phase. When changing the mobile phase, carefully consider the elution strength and separation selectivity3).
→ This is the best method if it is allowed to change analytical conditions.
- Reduce the ratio of methanol in the mobile phase. At the same time, select a column applicable to the analysis (shorter column with weaker retention).
→ However, abandoning an ODS column will sacrifice broad applicability and requires technical expertise.
- Stop using online degassing.
→ Stopping online degassing could cause problems with air bubbles in flow lines that might make quantitative precision worse or could prevent achieving stable detection1). Therefore, it is not recommended.
- Degas sample solutions before injection. Simply degassing with helium for about 10 seconds results in significant degassing.
→ However, the process adds complexity/time and may not be effective for continuous operations.
As a measure to improve separation of target components,
1) Change the elution position of ionic target components by changing the pH level of the mobile phase.
2) Change the type of organic solvent.
These measures should really be considered during method development.
As described above, even if some ghost peaks are judged to be caused by dissolved air, it may be difficult to resolve the problem easily. Nevertheless, it is extremely important for those developing and managing analytical conditions to understand the causes.
2) S.R. Bakalyar, M.P.T. Bradley and R. Honganen, J. Chromatogr., pp. 158, 277-293 (1978)
3) LCtalk vol. 35, pp. 8-9 (1995)