A buffer solution is an aqueous mixture designed to resist changes in acidity or alkalinity when small amounts of acid or base are added. It achieves stability by containing a weak acid and its conjugate base, which neutralize introduced ions. Degassing is the physical process of removing dissolved atmospheric gases, primarily nitrogen and oxygen, from the solution before use. This step maintains the integrity and precision of many analytical chemistry and biochemical applications.
Why Degassing is Essential for Laboratory Work
Dissolved gases in a buffer solution cause significant problems in high-precision laboratory techniques, particularly in analytical applications like High-Performance Liquid Chromatography (HPLC). These gases compromise data integrity and system performance by creating physical disruptions within the instruments. The most common issue is pump cavitation, which occurs when dissolved gas comes out of solution inside the pump head due to pressure changes. This formation of micro-bubbles causes flow rate inconsistencies, leading to erratic retention times and unreliable quantitative analysis.
Bubbles that pass through the system and into the detector cell scatter light, generating baseline noise or spurious peaks in the chromatogram. This interference makes accurate peak identification and integration difficult. Even outside of chromatography, dissolved gases can spontaneously form bubbles when a solution is drawn through a porous medium, such as a filtration column. These bubbles interfere with volumetric measurements, block flow paths in gel filtration columns, or corrupt readings from sensitive sensors.
Practical Procedures for Degassing Buffer Solutions
The choice of degassing method depends on the required level of gas removal, the buffer volume, and the sensitivity of the final application. For critical work, such as preparing mobile phases for ultra-high performance liquid chromatography, a combination of methods or the most efficient technique is used. The goal is to lower the concentration of dissolved gases below the saturation point to prevent bubble formation during the experiment.
Vacuum Filtration/Aspiration
Vacuum filtration is an efficient method because it simultaneously removes particulate matter and dissolved gas. The procedure involves pouring the buffer into a side-arm flask fitted with a vacuum-rated filter unit, often containing a 0.45 µm membrane filter. A vacuum pump is connected to the side arm, and a low vacuum is applied to the flask.
The reduced pressure above the liquid surface lowers the solubility of the dissolved gases, causing them to bubble out of the solution. Stirring the solution with a magnetic stir bar accelerates the release of gas. The solution is degassed until the visible stream of bubbles ceases, a process that can take up to an hour for larger volumes.
Ultrasonic Bath (Sonication)
Sonication involves placing the buffer solution in a flask within an ultrasonic bath, which uses high-frequency sound waves to promote degassing. The mechanical vibrations agitate the liquid at a microscopic level, causing dissolved gas molecules to coalesce and rise to the surface. This method is fast, typically requiring about 15 minutes of exposure.
Sonication is considered the least effective standalone method, often removing only 20 to 30 percent of the dissolved gases. While it is a quick option for routine buffers where moderate degassing is acceptable, it is insufficient for sensitive analytical work like chromatography. For critical applications, sonication is best used alongside vacuum or sparging to enhance gas removal efficiency.
Helium Sparging
Helium sparging is a highly effective offline degassing technique, capable of removing over 80 percent of dissolved gases. This procedure involves bubbling a stream of inert helium gas through the buffer solution using a specialized porous glass frit submerged in the liquid. The helium, which has low solubility, displaces the dissolved atmospheric gases (oxygen and nitrogen) as it rises through the solution.
The helium flow must be maintained at a low pressure to create fine bubbles without causing evaporation of volatile buffer components. Although highly efficient, continuous or prolonged sparging can alter the solution’s composition due to solvent evaporation. Sparging is typically performed for a short, controlled period, such as five minutes, to achieve the desired level of gas removal.
Maintaining the Degassed State During Use and Storage
Once a buffer solution has been degassed, it immediately begins to re-absorb atmospheric gases upon contact with air. Maintaining the degassed state requires careful handling and storage to ensure the solution remains suitable for use. The simplest measure is to store the buffer in a tightly sealed container with minimal headspace, reducing the area where gas exchange can occur.
Avoiding vigorous agitation or mixing of the solution is necessary, as this action quickly reintroduces air. Solutions intended for use in High-Performance Liquid Chromatography systems can be protected by placing a solvent reservoir cap over the bottle that vents through a solvent filter or a specialized check valve. In older or specialized systems, a low-flow stream of helium can be continuously passed over the surface of the solvent in the reservoir, creating an inert blanket that prevents re-absorption.
For long-term storage, keeping the buffer at a cool temperature (typically 5 to 10 degrees Celsius) helps slow down the kinetics of gas re-absorption. Buffers containing salts are susceptible to microbial growth and precipitation over time, even when stored correctly. It is recommended to prepare only the volume of degassed buffer needed for the immediate experiment or a short period of use, such as a few days, to minimize waste and storage time.