How to Achieve Optimal Mobile Phase Performance: 6 Key Points

Hello, everyone,

Today, I'm sharing with you

The filtration, storage, degassing,

and replacement precautions of the mobile phase.

Filtration of the mobile phase

All solvents must be filtered through a 0.45µm or 0.22µm membrane filter before use to remove impurities and particles. This applies even to chromatographic-grade solvents unless labeled as "pre-filtered." Acetonitrile that has been stored for a long time may need filtration due to the potential presence of dimers or trimers. When using membrane filtration, it is crucial to differentiate between organic phase hydrophobic filters and aqueous phase hydrophilic filters.

2. Storage of the mobile phase

The storage of the mobile phase should be based on the subsequent experiments, selecting appropriate storage containers. For liquid chromatography, glass solvent bottles are generally chosen, while for ion chromatography eluents, polypropylene eluent containers are preferred. The storage containers must be tightly sealed to prevent solvent evaporation, which can harm individuals, cause changes in composition, and prevent oxygen and carbon dioxide from dissolving into the mobile phase.

Phosphate and acetate buffer solutions are prone to mold growth and should be freshly prepared and used whenever possible, rather than being stored. Containers should be regularly cleaned, especially bottles containing water, buffer solutions, and mixed solutions, to remove impurities and possible microbial growth at the bottom.

3. Degassing of the mobile phase

The mobile phase used in HPLC must be degassed before use, otherwise, bubbles may escape into the system, affecting the pump's operation. Bubbles can affect detector sensitivity, baseline stability, and may even make detection impossible. Dissolved gases can also cause changes in the solvent's pH, leading to errors in separation or analysis results.

Dissolved oxygen in the mobile phase can react with samples, the mobile phase itself, and even the stationary phase such as alkylamines. Dissolved oxygen can form complexes with certain solvents such as methanol and tetrahydrofuran, which absorb UV light. In fluorescence detection, dissolved oxygen can cause quenching under certain conditions. In electrochemical detection, especially in reduction electrochemistry, the effect of oxygen is even more significant.

04 Common degassing methods

Offline degassing:

Methods such as ultrasonic degassing are used to remove larger bubbles from the mobile phase. Online degassing methods are used to remove smaller bubbles. Although many HPLC systems are now equipped with online degassers, ultrasonic degassing is still necessary. Online degassing can only remove relatively small bubbles, and it is difficult to remove large bubbles, leading to overloading of the degasser and affecting its lifespan.

Vacuum degassing (online degasser)

This is also a commonly used degassing method. The mobile phase passes through a degassing chamber under vacuum pressure, causing dissolved gases to form bubbles and overflow. Its effectiveness is second only to helium degassing. Currently, the vast majority of liquid chromatography systems use online degassers, namely vacuum degassing.

Other degassing methods:

Helium degassing: Can remove 80% to 90% of dissolved gases and effectively eliminate stubborn gases dissolved in aqueous solutions. It is commonly used in electrochemical detection. However, due to its high cost, it is not widely used in other experiments.

Heating and reflux degassing: Only suitable for mobile phases without organic solvents.

05 Solvent filters

Solvent filters for the mobile phase can prevent tiny particles from entering the HPLC system. Some filter heads have a stepped connection outlet at the top, which can be used for connecting pipelines with inner diameters of 1.5mm, 2.2mm, and 3.5mm, respectively. The filters should be cleaned regularly, especially those used with aqueous phases, which should be cleaned regularly and promptly.

06 Precautions for changing the mobile phase:

Before replacing the mobile phase, remove the solvent filter head from the solvent surface, shake off most of the remaining solvent on the filter head, and then place it into the next mobile phase to prevent the residual solvent from altering the next mobile phase.