Offline vs. Online Degassing of HPLC Solvents

Gas bubbles and outgassing can significantly impact HPLC performance. Outgassing occurs when the amount of gas exceeds what the HPLC solvent can absorb. The solvent's ability to absorb gas depends on factors like pressure, temperature gradients, and the specific liquid and gas involved. Rough surfaces within the HPLC system can also serve as nucleation sites for gas bubbles. The presence of gas bubbles in solvents can lead to shifting retention times, fluctuating baselines, unpredictable changes in peak areas, irregular peak shapes, and pump failures. These issues can result in inaccuracies and reduced precision.

Offline vs. Online Degassing of HPLC Solvents

Offline Degassing

Offline degassing is when the mobile phase is degassed before being placed in the HPLC system. Offline degassing is commonly done through helium sparging. Helium sparging uses helium that is bubbled through the mobile phase to dissolve and displace air and contained in a slightly pressurized mobile phase reservoir bottle. It is recommended to use a solvent inlet filter such as a 2um filter to prevent particulates originating from the sparging system from entering the mobile phase reservoir and to help disperse the sparging gas efficiently. However, offline degassing holds a major disadvantage in comparison to online degassing, as the mobile phase will begin to reabsorb air when it is placed in the HPLC system. For this reason, it is important to have slight back pressure throughout the entire system, as bubbles will reform at atmospheric pressure. A back pressure regulator can be installed after the detector, to minimize bubble formation (check your detectors manual for flow cell pressure rating).

Online Degassing

In online degassing, degassing occurs during analysis to eliminate the reabsorption of air. Online degassing is timesaving in the long run as it is more effective in improving reliability in results. Using a Vacuum degasser is the most common online degassing method. In online vacuum degassing, the degassing device is placed within the HPLC system just before the mobile phase enters the pumps. The device uses a gas-permeable membrane to extract nitrogen and oxygen molecules out of the mobile phase, allowing the mobile phase to be degassed as needed throughout the analysis. Outgassing can occur when pressure drops after the column. A back pressure regulator can be installed after the detector, to minimize bubble formation (check your detectors manual for flow cell pressure rating).

Offline and online degassing of HPLC solvents both use similar methods at different points in the mobile phase. Offline degassing offers many quick and simple degassing methods but runs the risk of reabsorbing air once solvents are placed in the HPLC system. Online degassing with a vacuum degasser has become industry standard for HPLC and typically requires little to no maintenance, contact Chrom Tech if you would like to add a vacuum degasser to your system. You can use these degassing methods for more reliable results. It’s also important to remember that HPLC solvents are considered a hazardous waste product by the FDA, and solvent waste containers should be used for safe HPLC waste collection.