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📅 Last Updated: October 24, 2026
Degassing of HPLC mobile phase removes dissolved air so bubbles do not form when solvents mix. Bubbles cause false peaks, noisy baselines, lower pump flow rate precision and possible pump failure. Helium purging, vacuum degassing and sonication are the common methods, and combining them works better than using one alone.
Why is degassing of HPLC mobile phase done? Degassing of HPLC mobile phase is the removal of dissolved air from the solvents before or during analysis. This stops bubbles from forming in the pump, tubing or detector. When different liquids mix in HPLC analysis, bubbles can cause problems. To avoid this, analysts remove dissolved air by degassing the liquid used in the analysis.
Unstable and Noisy Baselines: Degassing of HPLC Mobile Phase
Degassing is a step that removes dissolved air from the liquid used in HPLC analysis. Air bubbles hurt the pump and the detector signal. This section explains how the problem arises and how mixing type changes it.
Bubbles in the piston chamber cause cavitation and affect check valve performance, which lowers the pump’s flow rate precision. Excessive pressure can develop and lead to eventual pump failure. Schedule M and 21 CFR Part 211 expect laboratory instruments to be suitable for their use. A pump with cavitation or a faulty check valve from dissolved air is therefore a GMP concern. Regular calibration of HPLC also supports pump performance.
When air bubbles go through the detectors, they create false peaks. Bubbles disturb the liquid flow through the HPLC tubing and create gaps in the flow, which disturbs the detector signal. Under ICH Q2(R2), an analytical procedure must show suitable accuracy, precision and specificity, and false peaks or an unstable baseline undermine all three. Baseline noise also affects system suitability in HPLC, and false peaks can lead to false OOT results.
First, here is how the problem arises. Solvents equilibrate with atmospheric gases on exposure to the laboratory environment. On mixing solvents, the solubility of air is lower than in the same proportion of pure solvents, so excess air bubbles out.
When liquids mix or when there are rough surfaces in the system, bubbles might form. You do not need to remove all the air. Reduce it below the level at which bubbles form. Systems that mix at low pressure tend to make more bubbles. With low-pressure mixing, the liquids are blended at regular air pressure, and bubbles can form anywhere along the flow.
With high-pressure mixing, the liquids are mixed after going through pumps and are blended in a high-pressure chamber. Even if the mixture is full, the high pressure stops bubbles from forming. If there are any bubbles, they might show up when the liquid comes out of the column and goes back to normal air pressure.
HPLC Degassing Techniques
Three methods are in common use for the HPLC mobile phase. Heating is also possible but not recommended.
Commonly used degassing practices for the HPLC mobile phase are:
- Helium purging
- Vacuum degassing
- Sonication
Each method removes a different share of the dissolved air:
- Helium purging removes about 80%.
- Vacuum degassing removes more than 60%.
- Sonication alone removes about 30%.
Heating is the best way to completely remove air from a liquid. It is not recommended, because it can remove components of the mobile phase along with the air. It also takes a long time to bring the liquid to the right temperature.
1. Helium Pursing:
Helium can remove about 80% of the air in the liquid. For a mix of water and certain chemicals, using the same amount of helium as the liquid works well. Don’t use too much helium, because it can remove components of the mobile phase.
2. Vacuum degassing:
Vacuum degassing can take out more than 60% of the air. One way is to pull the liquid through a special filter with a vacuum. Many commercial systems do this while the liquid is still in the machine. The special tubing inside removes the air but keeps the liquid inside. USP <621> Chromatography sets the system requirements and adjustment limits for liquid chromatography, so the mobile phase must be bubble-free for the system to meet them.
3. Sonication:
Ultrasonic baths are quite common in labs for sonication. Used alone, sonication eliminates only around 30% of the air dissolved in the liquid. Combine it with other techniques for better results.
Remove air from the mobile phase in both low and high-pressure mixing setups. Combining methods reduces bubble problems more than any single method.
Many systems available for purchase come with on-line vacuum degassing. When you use sonication alongside this on-line degassing, you can achieve good outcomes.
Frequently Asked Questions
Three practices are commonly used: helium purging, vacuum degassing and sonication. Helium purging removes about 80% of the dissolved air, vacuum degassing more than 60%, and sonication alone about 30%. Many commercial systems also come with on-line vacuum degassing, and you can use sonication alongside it for good outcomes.
Used alone, sonication eliminates only around 30% of the air dissolved in the liquid. Ultrasonic baths are quite common in labs, but this figure is lower than for helium purging or vacuum degassing. For better results, combine sonication with other techniques, such as on-line vacuum degassing.
Yes. Systems that mix at low pressure tend to make more bubbles, because the liquids blend at regular air pressure and bubbles can form anywhere along the flow. High-pressure mixing stops most bubbles from forming. Even so, removing air from the mobile phase is practical in both low and high-pressure mixing setups.
Heating is the best way to completely remove air from a liquid. It is not recommended because it can remove components of the mobile phase along with the air. It also takes a long time to bring the liquid to the right temperature. Helium purging, vacuum degassing and sonication are the usual choices instead.
No. You do not need to remove all the air. Reduce it below the level at which bubbles form. Bubbles can form when liquids mix or when there are rough surfaces in the system. Combining methods reduces bubble problems more than any single method.
References
- Schedule M, CDSCO, Drugs Rules, 1945
- 21 CFR Part 211, US Code of Federal Regulations (eCFR)
- ICH Q2(R2), International Council for Harmonisation (ICH)
- USP <621>, United States Pharmacopeia (USP–NF)
Pankaj Sharma — Quality Control Specialist
Pankaj Sharma is a pharmaceutical QC professional who reviews our technical content for GMP accuracy, laboratory practices, and regulatory relevance.