Reasons for Degassing Liquid and What to Consider
All solutions equilibrated at room temperature and pressure will contain dissolved gases. Sample degassing is the process of removing these dissolved gases from the solution. Removal of these gases may be necessary because the gases may escape from the solution and form air bubbles in your syringe, which can negatively affect the performance of your syringe pump. Additionally, removal of these gases could prevent unwanted side reactions, such as redox reactions, in your samples.
When performing microfluidic experiments, you should be especially mindful of air bubbles. In these experiments, the presence of air bubbles can cause flow rate instability, increase the time required for pressure equilibration, and increase fluidic resistance1. If you are working with cell culture, air bubbles can rupture cell membranes when the surface tension of the air-liquid interface is sufficiently large21. In applications involving proteins, air bubbles can promote protein unfolding and subsequent aggregation by reducing the hydrophobic effect3.
Here are four common types of liquid degassing techniques that you may consider prior to loading your syringe.
1. Degassing Using Helium Sparging
In this method, helium gas is slowly and continuously bubbled through a sample for 5 minutes, displacing dissolved gases within the sample4. As an inert gas with low solubility in water5, helium will not react with solutes within the sample nor will it significantly dissolve within the sample.
Helium sparging is less effective than other pump degassing methods, however, it is quick, easy and applicable to many sample types.
2. Degassing Using a Vacuum
Degassing samples using a vacuum is one of the most commonly used laboratory methods. Vacuum pump degassing is effective because of the phenomenon described by Henry’s law, which states that the amount of dissolved gas is proportional to its partial pressure in the gaseous state.
Introduction of a vacuum above your sample will reduce the partial pressure of air and hence reduce the solubility of dissolved gases within the sample. Degassing using a vacuum can be achieved by slowly stirring the sample for 5 minutes with a magnetic stir bar in a sealed filtering flask connected to a vacuum pump or water aspirator. If the volume of the sample is too low for a vacuum flask, the sample can be placed in a small open glass vial and sealed in an evacuated vacuum desiccator.
It is important to note that sample evaporation will be enhanced under vacuum and thus, if your experiments are highly sensitive to sample concentration, it might be necessary to determine the sample concentration after degassing, especially for small volume samples.
3. Degassing Using Sonication
Sonication is a widely used technique to degas solutions. In sonication, propagating sound waves cause alternating high and low pressure cycles (6). These low pressure cycles create vacuum bubbles that trap dissolved gas. The sonication waves cause smaller vacuum bubbles to coalesce and rise to the surface, where it releases the entrapped gas to the environment.
The effectiveness of sonication can be enhanced by applying low to moderate amplitudes, using sonotrodes with large surface area and heating the liquid6.
Sonication is often combined with the vacuum method (see above), where it effectively replaces magnetic stirring for enhanced release of air from the sample4. If the samples contain live cells, care should be exercised as sonication can lyse cell membranes.
For protein samples, excessive sonication can, in some cases, promote aggregation. Hence, researchers may have to adapt sonication parameters to suit their experimental system.
4. Degassing Using Freeze-Pump-Thaw
Freeze-pump-thaw is a highly effective degassing method that can be used for low volume samples. In a typical experiment, the sample is placed in a Schlenk flask and frozen using liquid nitrogen7. The headspace above the sample is then evacuated using a vacuum pump, which lowers the solubility of dissolved gas within the liquid according to Henry’s law (see above). The sample is then sealed and thawed, allowing dissolved gases to migrate into the evacuated headspace.
This process can be repeated once or twice to enhance degassing efficiency. Similar to the sonication method, the freeze-pump-thaw method may result in cell lysis and/or protein aggregation and thus may not be appropriate for all sample types.
Sample degassing is important for reducing the possibility of air bubble formation, which can reduce pump performance, and negatively affect your microfluidic experiments. Depending on your sample type, you can consider using helium sparging, vacuum, sonication, and freeze-pump-thaw methods, either individually or in combination, to achieve degassing.
- AIR BUBBLES AND MICROFLUIDICS: TIPS AND TRICKS TO REMOVE THEM. Elveflow. [Online] December 29, 2017. http://www.elveflow.com/microfluidic-tutorials/microfluidic-reviews-and-tutorials/air-bubbles-and-microfluidics/.
- Systematic Prevention of Bubble Formation and Accumulation for Long-Term Culture of Pancreatic Islet Cells in Microfluidic Device. Yong Wanga, Dongyoung Leea, Lisa Zhanga, Hyojin Jeona, Joshua E. Mendoza-Eliasa. 2012, Biomed Microdevices, pp. 419-426.
- Protein structural dynamics at the gas/water interface examined by hydrogen exchange mass spectrometry. Yiming Xiao, Lars Konermann. 2015, Protein Science, pp. 1247-1256.
- Painter, Tammie. Methods for Degassing Buffers. Sciencing. [Online] December 29, 2017. https://sciencing.com/methods-degassing-buffers-6925709.html.
- Gevantman, L. H. SOLUBILITY OF SELECTED GASES IN WATER. [book auth.] William M. Haynes. CRC Handbook of Chemistry and Physics. Boca Raton, FL : CRC Press (Taylor and Francis Group), 2010, pp. 8-80-8-83.
- Ultrasonic Degassing and Defoaming of Liquids. Hielscher – Ultrasound Technology . [Online] December 29, 2017. https://www.hielscher.com/degassing_01.htm.
- JoVE Science Education Database. Organic Chemistry. Degassing Liquids with Freeze-Pump-Thaw Cycling. Cambridge, Massachusetts, United States : s.n., 2017.