Introduction

Mobile phase quality is one of the most important factors affecting chromatographic performance. Even when high-purity solvents are used, exposure to the atmosphere allows gases to dissolve into the mobile phase over time.  Common dissolved gases include:

• Oxygen (O₂)
• Carbon dioxide (CO₂)
• Nitrogen (N₂)
• Atmospheric air

Although often present at low concentrations, these gases can affect chromatography when they come out of solution within the HPLC system.  For this reason, degassing is considered a standard best practice in HPLC operation.

How Gases Enter Mobile Phases

Most chromatography solvents readily absorb gases from the surrounding environment.  This can occur when:

• Solvent bottles are left open
• Mobile phases are mixed and stored
• Reservoirs are repeatedly accessed
• Solvents are exposed during preparation

Even a freshly prepared mobile phase can begin absorbing atmospheric gases shortly after preparation.

Why Dissolved Gases Matter

Under low-pressure conditions, dissolved gases remain in solution. As solvents pass through pumps, mixers, detectors, and columns, changes in pressure and temperature may cause gases to come out of solution.  When this occurs, small bubbles may form within the fluid path.  These bubbles can interfere with normal system operation and affect chromatographic performance.

Effects on Pump Performance

HPLC pumps are designed to move essentially incompressible liquids.  When gas bubbles are present:

• Pump efficiency may decrease
• Flow delivery may become inconsistent
• Pressure fluctuations may occur
• Retention times may vary

Because gases are compressible while liquids are not, system performance can become less predictable when significant amounts of dissolved gas are present.

Effects on Retention Times

Stable flow delivery is essential for reproducible chromatography.  When dissolved gases influence pump performance, analysts may observe:

• Retention time drift
• Run-to-run variability
• Reduced reproducibility
• Inconsistent method performance

These effects may be particularly noticeable in highly sensitive analytical methods.

Effects on Baseline Stability

One of the most common symptoms of insufficient degassing is increased baseline noise.  Potential effects include:

• Baseline spikes
• Baseline instability
• Detector noise
• Signal fluctuations

These disturbances can make peak integration more difficult and reduce confidence in analytical results.

Detector Issues Associated with Gas Bubbles

Gas bubbles entering detector flow cells can create significant analytical problems.  Possible symptoms include:

• Negative peaks
• Positive spikes
• Unstable baselines
• Irregular detector response
• Reduced sensitivity

These effects are often more severe at lower analyte concentrations where signal quality is particularly important.

Special Considerations for Polymeric Columns

Some chromatographic columns are more sensitive to the effects of dissolved gases than others.  Polymeric stationary phases, in particular, can be more susceptible to performance issues associated with outgassing and bubble formation.  For these columns, careful solvent preparation and effective degassing can be especially beneficial.

Common Degassing Techniques

Several methods are commonly used to remove dissolved gases from mobile phases:

Vacuum Degassing

Vacuum filtration units can remove both particulates and dissolved gases from solvents prior to use.

Helium Sparging

Helium sparging displaces dissolved atmospheric gases and has long been used in chromatography laboratories.

Ultrasonic Degassing

Ultrasonic treatment can help release dissolved gases from solution.

In-Line Vacuum Degassers

Many modern HPLC systems include integrated degassers that continuously remove dissolved gases during operation.

Maintaining Degassed Solvents

Removing dissolved gases is only part of the process.  Once degassed, solvents should be protected from unnecessary atmospheric exposure.  Recommended practices include:

• Keeping solvent reservoirs covered
• Minimizing open-bottle exposure
• Using clean solvent containers
• Preparing fresh mobile phases when appropriate
• Maintaining degasser performance

These practices help preserve solvent quality for longer periods.

Benefits of Proper Degassing

Effective mobile phase degassing can help provide:

• Improved baseline stability
• Better retention time reproducibility
• Reduced detector noise
• More reliable pump performance
• Improved quantitative accuracy
• Greater system reliability

These benefits are particularly important for regulated, validation, and trace-level analytical methods.

Applications

Careful mobile phase degassing is recommended for:

• HPLC
• UHPLC
• LC-MS
• Gradient methods
• Isocratic methods
• Pharmaceutical analysis
• Environmental testing
• Food and beverage analysis
• Biological sample analysis

Virtually all liquid chromatography applications can benefit from proper solvent preparation and degassing practices.

Conclusion

Mobile phases naturally absorb atmospheric gases such as oxygen and carbon dioxide during preparation and storage. If these gases are not removed, they can contribute to pump instability, baseline noise, detector disturbances, and retention variability. Proper degassing and solvent handling practices help maintain consistent chromatographic performance, improve system reliability, and support accurate analytical results.