Have You Paid Attention to These Things With Mobile Phase?

The mobile phase takes too long, but it will directly affect the results of your experiment. Here Hawach will talk to teachers about the precautions for the mobile phase.

One: storage of mobile phase

The mobile phase is generally stored in glass, polytetrafluoroethylene, or stainless steel containers, and cannot be stored in plastic containers. Many organic solvents, such as methanol and acetic acid, can leach the plasticizer on the plastic surface, causing the solvent to be contaminated. Such contaminated solvents, if used in HPLC systems, may cause flash column efficiency to decrease. The storage container must be tightly covered to prevent changes in the composition caused by solvent volatilization, and to prevent oxygen and carbon dioxide from dissolving into the mobile phase.

Phosphate and acetate buffers are prone to mildew, so they should be prepared as freshly as possible and not stored. If it is necessary to store it, it can be stored in the refrigerator and re-filtered before use. The container should be cleaned regularly, especially the bottle containing water, buffer, and mixed solution, to remove the impurities at the bottom and the possible growth of microorganisms.

sax chromatography flash column

Two: The influence of mobile phase storage time

01. Organic phase volatilization: the greater the composition ratio of the organic reagent, the lower the boiling point, and the greater the degree of volatilization, but the volatilization is relatively well controlled, as long as the seal is good;

02. Influence of pH value: The mobile phase of reversed-phase chromatography is often a buffer salt, and its pH value is affected by the volatilization of the organic phase, carbon dioxide in the air, etc., sometimes even turbid; and different pH values ​​and different buffer salts have different effects;

03. Microbial influence: The water phase system for a long time will often cause microorganisms, especially affected by light, so it is often stored in a brown bottle; in addition, if the organic phase ratio in the mobile phase is larger, it is less likely to cause growth Microorganisms; therefore, the validity period varies according to storage conditions and organic phase ratios.

So teachers should pay attention. It is recommended that the liquid mobile phase be replaced with the bottle after 12 hours, the pure organic phase is recommended to be filtered in 12 hours, and the bottle should be replaced after 24 hours. The mobile phase solution is most resistant to microbes exceeding the standard. Various bacteria can cause the column to block up and cause many adverse effects.

We all know that if the room temperature is high, there may be microbial reproduction in the morning and the afternoon when the pure water phase is prepared, and it needs to be re-filtered. Therefore, the pure water phase should be prepared for immediate use. Don’t be lazy and unfit for luck. If the result is large, you may rework it at any time.

Three: Filter and degas the mobile phase before use

Before using the mobile phase, filter and degas with a 0.45μm filter membrane to remove particles and dust. In the liquid feeding pump, in order to prevent impurities (solids) from entering the flow path, a suction filter is installed, but the mesh pore size is 10μm, and particles smaller than this can still pass, which will cause blockage and pollution of the flow path and the column.

For this reason, it is recommended to use 0.45μm membrane filtration. The mobile phase solvent must be degassed. If it is not degassed, a large number of bubbles will be easily generated when the solvent is mixed or the pressure and temperature change, which will cause the pump to malfunction, the infusion is not smooth, and the detector produces noise signals.

Four: The influence temperature

Monitoring and controlling the temperature of the mobile phase is crucial in chromatography. It affects viscosity, density, solubility, and retention times of analytes. Proper temperature control ensures accurate and reproducible results. It’s particularly critical in techniques like gas chromatography, where temperature plays a pivotal role. Additionally, extreme temperatures can impact column stability and analyte integrity. Overall, considering and regulating temperature is fundamental for successful chromatographic separations.