Liquid Chromatography – Dissolved Air and Peaks

Part 1. Introduction

Sometimes when checking chromatograms, we notice peaks of unknown origin, so called ghost peaks. We already touched on the subject of ghost peaks in the 5th edition of our LC blog when we discussed the water quality.

Additionally, unknown peaks can be particularly troublesome when testing pharmaceuticals for impurities, both known and unknown impurities. There are various causes of ghost peaks but one possible cause that is often neglected is dissolved air in the sample solvent. This month we will focus on the dissolved air and its impact on chromatography. Before we progress further let’s make some assumptions, we will focus on reversed-phase separation with UV detection below.

Part 2. Dissolved air (Oxygen) and appearance of unknown peaks

In some cases when the type and composition of the organic solvent between the sample solution and the mobile phase in bottles is different, it can be predicted that some additional peak(s) in chromatograms will appear. However, we can’t neglect the fact that there are also cases where peaks appear in the blank injection of the mobile phase. One possible cause of this is a difference in the amount of dissolved air or, particularly with UV detection, dissolved oxygen between the mobile phase in bottles on top of the system and the mobile phase in the vial, injected as blank. Figure 1 below compares the chromatograms obtained by injecting the mobile phase blank. Mobile phase was a mixture of 85/15 of Methanol / Water. This was vialed but also used as mobile phase on the HPLC system and of course degassed online.

Figure 1. Mobile Phase blanks with various Oxygen saturation levels – HPLC Mobile Phase was DEGASSED

Each injection in the Figure 1 above was carried out with mobile phase in vials treated differently to either add or remove the saturated oxygen from the liquid in vials. When the sample (mobile phase) was injected in the normal state, chromatogram (a) in Figure 1, a peak at approx. 2.7min appeared with around 10 mAbs intensity. It is interesting that the same peak nearly disappears when the sample (mobile phase) was sparged with helium and injected. The concentration of dissolved oxygen is close to zero; see chromatogram (b) in Figure 1. On the other hand, the peak at 2.7min reached nearly 60mAbs intensity when the sample (mobile phase) was injected immediately after purging with oxygen. Refer to Figure 1 chromatogram (c). All chromatograms obtained in Figure 1 were run on system where Mobile Phase (HPLC bottles) was degassed online.

What is fascinating is that when the mobile phase used on HPLC system was not subjected to online degassing, hardly any peak appeared when the samples (mobile phase blanks) were injected. When the injected sample (mobile phase) was in the normal state, chromatogram (a) in Figure 2, no peaks at approx. 2.7min were observed. In the injection of Helium purges sample (mobile phase blank) a negative peak is present at 2.7min, indicating that the oxygen dissolved within the sample was replaced by Helium; see chromatogram (b) Figure 2 below. Then again, the peak at 2.7min reached nearly 50mAbs intensity when the sample (mobile phase) was injected immediately after purging with oxygen. Refer to Figure 2 chromatogram (c).

Figure 2.Mobile Phase blanks with various Oxygen saturation levels – HPLC Mobile Phase NOT DEGASSED.

Based on the above experiment it can be concluded that differences in the amount of dissolved oxygen in the mobile phase and in the samples (mobile phase blanks in our case) can result in the appearance of peaks with various intensities. Additionally, degassing or not can have an impact on the chromatographic profile.

Part 3. Peaks Size

Let’s discuss how easily you can estimate a peak size based only on the information obtained from UV spectra. Figure 3 shows a comparison of UV spectra obtained for Methanol which was both degassed and air saturated (i.e., not degassed). When methanol is degassed, the absorbance is clearly reduced. Knowing that methanol absorbs nearly up to 260 nm the differences in absorbance of degassed vs not degassed methanol will depend on the wavelengths compared. At lower wavelengths the difference in absorbance (i.e. 210 nm) exceeds 300 mAbs (0.3 AU) whereas at higher wavelengths (i.e. 254 nm), the difference can nearly be neglected as is at the level of 10 mAbs (0.01 AU). Using these values, a simple calculation can be performed to obtain estimated peak height for the case where air-saturated methanol is injected into degassed methanol mobile phase.

Figure 3. Comparison of Methanol UV spectra – Degassed and NOT Degassed (air saturated).

Assuming flow rate of 1 mL/min and an injection volume of 10 uL, if we view the peak as a triangle with the base corresponding to period of 0.4 minutes, we can estimate from the Figure 3 that the peak height at 210 nm exceeds 15 mAbs (0.15 AU) and the peak height at 254 nm is approx. 0.5 mAbs (0.05 AU). The estimates are simply the difference at 210nm and 254nm between the degassed and not degassed methanol lines. We can clearly see that the peaks are quite large at short wavelengths, as expected.

The case above was only considering the methanol as solvent but what about other solvents? Figure 4 shows differential UV spectra for various types of solvent, obtained by subtracting the spectra for the degassed state from spectra for the air-saturated state for each of the solvents presented in Figure 4. The absorbance of each of the solvents is increased by the dissolved air. The influence of dissolved air is small for water and acetonitrile and has much larger impact for hexane, methanol, and tetrahydrofuran (THF). All solvents were HPLC grade solvents.

Figure 4. Differential UV spectra for various solvents – spectra obtained by subtraction of degassed and NOT Degassed (air saturated) UV spectra for each of the solvents.

The changes in absorbance observed in Figure 4 do not correspond to the solvents’ oxygen solubility levels/capabilities. For example, hexane has a much higher oxygen solubility level than methanol¹⁾ but the change in absorbance for hexane is much smaller than the one for methanol. We can therefore conclude that the absorbance is impacted by the interaction between oxygen and the solvent, rather than the absorbance of oxygen itself.

Part 4. Dissolved air (oxygen) peaks elution order

There can be an incorrect assumption that peaks originated from dissolved oxygen should elute early, close to solvent front. Like the retention behavior of the sample constituent, elution of dissolved oxygen occurs later if the organic content of the mobile phase is decreased (i.e. methanol). Figure 5 below shows an example of elution in which the mobile phase and injected blank (mobile phase) have the same composition, i.e. for 3/7 Mobile Phase ration a 3/7 blank was injected and so on. It is clear that the peak shifts with the increase / decrease of organic content within the mobile phase. The peak becomes smaller as the proportion of methanol becomes lower.

This tendency is also evident when phosphate buffer solution is used as mobile phase instead of water. We must remember that with these elution profiles there is a risk that coelution with the sample compound (component) may occur.

Figure 5. Elution of dissolved air peak impacted by Mobile Phase composition

Part 5. How to investigate if the unknown peak originates from dissolved air

How to investigate if the unknow peak is actually originating from dissolved air? You can use the following guideline to estimate the suspicious peak that appears while using a gas-liquid separation membrane online degasser in your HPLC system. If the below criteria are satisfied, there is a high possibility that the peak originated in dissolved air.

• Unknown peak presence – in all injections peak is present with approx. the same intensity and at approx. the same retention time when injection volumes are the same and all samples / standards are diluted with the same sample solvent.
• Check for increased peak size when vial is saturated with air – open the vial and agitate (shake) the vial or leave the vial in a semi-open system in order to saturate the liquid in vial with additional air. When the sample solvent is diluted and “X” times the volume is injected, approx. “X” times the area of the unknown peak is obtained.
• Inject mobile phase blank – check if the peak in mobile phase blank injection elutes at the same/similar retention time as suspected (unknown) peak in your samples/standards injections. When mobile phase is injected after degassing (by purging with helium for about 10 s), the peak should be smaller or not present.
• Omit the degasser – when the mobile phase is delivered without passing it through a degasser, and the sample solution is injected, the peak becomes smaller. If mobile phase blank is injected, the peak definitely becomes smaller than it was in the point above when compared. It may not necessarily become smaller, however, if the sample-solvent composition is different to the mobile phase composition present in the bottles on the HPLC system.

Always aim to compare like with like with the minimum deviation.

Part 6. Countermeasures and Summary

You can already see that it is difficult to completely remove peaks originated from dissolved air, but there are few steps which can help to reduce them. Below statements are true when assumed that the mobile phase used was a mix of a methanol and water.

1. Replace the methanol in mobile phase with acetonitrile, if possible. When replacing the organic constituent of mobile phase consider the elution capacity and the separation selectivity. This is the best approach if such a change is possible. Some method once validated are locked and this type of change will not be allowed.
2. Lower the proportion of methanol in the mobile phase, if possible. Again, the same as in point 1 above – such a change might not be allowed.
3. Replace the column with one that is suitable for analysis, i.e., a column with low retention or a shorter column. Understanding of column interactions is a key for any chromatography and technical knowledge in such a change is critical.
4. Assess if online degassing will have a significant impact on the dissolved air peak presence. If required, stop online degassing of the mobile phase. However, based on my own experience this method is not recommended because the generation of bubbles in the flow line may adversely affect the quantitative accuracy and stable detection may not be possible.
5. Degas the sample solution before injection. I must make this point very clear – DEGAS SAMPLE Solution not the mobile phase offline – this can be substantially degassed by purging with helium for approx. 10sec. However, a lot of effort is required, and this method may not be very effective for continuous operation especially in the labs with high sample throughput.

In such a case where coelution of dissolved air peak and your main peak of interest occurs the following methods can be used to improve separation:

1. If the target constituent is ionic, change the elution position of the target constituent by slightly changing the pH value of the mobile phase within the allowed ranges.
2. Change the type or amount of organic solvent used – slightly improve gradient steps or isocratic composition without impact the validated state of your test method.

The above aspects should be generally considered and tested as part of method development and validation exercises to ensure robust and scientifically sound method is progress to QC labs for routine testing.

As described above, even if a ghost peak is somehow judged to originate from dissolved air, there may be many cases where the problem cannot be solved easily. Even so, and effort to determine the cause of the peak is extremely important for the personnel developing and controlling the analytical method conditions.

1) S.R. Bakalyar, M.P.T. Bradley and R. Honganen,, J. Chromatogr ., 158, 277-293 (1978)

Liquid Chromatography – Master the Basics

This article is part of our “Liquid Chromatography – Master the Basics” series, your go-to resource for comprehensive and insightful updates on the world of liquid chromatography. Each month in 2024 we will dive into a Liquid Chromatography topic, offering content that is both accessible to beginners and beneficial for experienced scientists.

Explore the Series