Mobile phase | shelf life

Do you know the shelf life of mobile phase? We often meet the question like “how long can we keep the mobile phase? Is it ok to keep it for a week?” But, do you know if the time of mobile phase is too long, but it will directly affect your experimental results? Here is a mobile phase information that needs to stay in your attention.

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 plasticizers on the plastic surface, resulting in solvent pollution. If the contaminated solvent is used in the HPLC system, the column efficiency may be reduced. The storage container must be tightly covered to prevent the composition change caused by solvent volatilization and prevent oxygen and carbon dioxide from dissolving into the mobile phase.

Phosphate and acetate buffers are easy to mold, so they should be prepared as fresh as possible and not stored. If it is really necessary to store, it can be refrigerated in the refrigerator and re filtered before use. Containers should be cleaned regularly, especially bottles containing water, buffer solution and mixed solution, to remove impurities, sediment and possible microorganisms at the bottom.

Two: Influence of mobile phase storage time

1. Volatilization of organic phase: the larger the proportion of organic reagents, the lower the boiling point, the greater the degree of volatilization, but the volatilization is relatively easy to control, as long as it is well sealed;

2. Impact 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 organic phase and carbon dioxide in the air, and sometimes even turbid; And different pH values and buffer salts have different degrees of influence;

3. Microbial influence: the water phase system will often cause microorganisms after a long time, especially affected by light, so it is often stored in brown bottles; In addition, the larger the proportion of organic phase in the mobile phase, the less likely it is to cause microorganisms; Therefore, the shelf life is different according to the storage conditions and the proportion of organic phase.

Therefore, we should pay attention. It is recommended to replace the liquid phase mobile phase with the bottle after 12 hours, and the pure organic phase with the bottle after 24 hours. The mobile phase solution should not exceed the standard of microorganisms. Various bacteria will lead to column plugging and produce many adverse effects. As we all know, if the room temperature is high, microorganisms may reproduce in the pure water phase in the morning and after work in the afternoon, so it needs to be re filtered. Therefore, the pure water phase should be prepared and used now. Don’t take chances and be lazy. If the result error is large, you may rework at any time.

Three: The mobile phase should be filtered and degassed before use

Filter and degassing the mobile phase with 0.45μm filter membrane before use to remove particles and dust. In the feed pump, suction filters are installed to prevent debris (solids) from entering the flow path, but the mesh aperture is 10μm, smaller particles can still pass through, which can lead to blockage and contamination of the flow path and column. Therefore, it is recommended to use 0.45μm membrane filtration. The mobile phase solvent must be degassed. If it is not degassed, it is easy to produce a large number of bubbles when the solvent is mixed or when the pressure and temperature change, which will lead to pump misoperation and poor infusion, and the detector produces noise signals.

Four: The mobile phase needs to be restored to room temperature for use

Use the mobile phase after returning to room temperature. When the mobile phase temperature is different from room temperature, it is difficult to stabilize the baseline level before the mobile phase returns to room temperature, especially drift, and bubbles are easy to occur. When water is mixed with organic solvents, the mixture varies greatly and often differs greatly from room temperature, so be careful.

Five: Avoid using corrosive mobile phase

The mobile phase contains concentrated sulfuric acid, concentrated nitric acid, dichloroacetic acid, dichloromethane, acetone, tetrahydrofuran, dimethyl sulfoxide and other solvents, which will deteriorate the strength of PEEK resin pipe in the flow path and easily break PEEK resin pipe to make the solvent spill out. Solvents containing halogen ions in the mobile phase, such as KCl, NaCI, NH4CI, etc., have corrosive effect on stainless steel materials in the flow path, so the above solvents should be avoided as far as possible. If necessary, the whole flow should be cleaned with distilled water immediately after the analysis.

Six: Measure pH value after adding organic reagent in mobile phase

The so-called pH value is based on water as a solvent system called pH value, so the correct way to adjust the pH value of buffer solution is carried out in an aqueous solution. If the pH value is measured after the organic solvent is added, it will actually be different from the pH value measured before the addition.

The most important thing, however, is consistency. If the custom is to measure pH after adding organic solvents, just make sure you do it every time. It’s not 100% accurate, but at least it’s consistent with what was done before. This may be more important for a complete study to be consistent than if you measure the pH before adding organic solvents to get accurate data once.

Seven: do not completely use up the mobile phase in the mobile phase reagent bottle

In addition to the risk of pump and column transport, there are other problems if the mobile phase is not adequately prepared. For example, the composition of the mobile phase at the top of the reagent bottle has changed because the water in the solution will evaporate from the top of the reagent bottle. If you use the last part of the bottle, which in fact is the top part of the original bottle, you can get residue trapped in the column as they pass through the column.

Eight: Degassing the mobile phase using ultrasound

The ultrasonic method is effective for ensuring that the buffer salts used are completely dissolved, but it is not the most efficient method for degassing, and due to the rapid heating of the solution during ultrasound, the organic reagent in the mobile phase evaporates. To save time, the mobile phase can be filtered through a vacuum filter for 5 minutes, which can achieve both degassing and filtration in one step.

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