High-performance liquid chromatography filter manipulation points

Any particulate matter entering the HPLC system is blocked by the screen at the inlet end of the column. The final result is a blockage of the column, which is characterized by an increase in system pressure and deformation of the chromatographic peak. Therefore, various precautionary measures must be taken, including the manipulation steps and various filtering designs of commodity instruments, in an effort to prevent or reduce the intrusion of particulate matter into the HPLC system, thereby prolonging the service life of the instrument and the column and improving the reliability of the data. In HPLC systems, there are three main sources of particulate matter: the active phase, the sample under test, and the wear of the instrument system components.

1, the measured sample

The second source of particulate matter in the liquid phase system is the sample under test. Before some laboratories placed their samples on autosampler trays (or manual injections), all samples were first filtered through a 0.45 μm syringe filter. This is a method of effectively removing particles in the sample under test. However, there is one point to be concerned about this process: If you use a syringe filter, it is not possible to obtain 100% of the measured sample that passes through the filter, and there will always be more or less loss. The loss comes from several aspects: the adsorption of the filter membrane, the adsorption of particulates from the filter, the leakage of the syringe filter and the syringe connection. If there is a loss, the content or concentration of the analyte in the filtered liquid is the same as the content or concentration of the original basic sample solution? This topic generally needs to be confirmed by experiment. Confirm this step is to increase the workload and cost. The use of filters is a kind of consumption, and the price of each filter ranges from a few dollars to more than ten dollars. However, in the analysis of residue in foods, the filtration step has become an indispensable step because of the complexity of the matrix. In the actual analysis work, each sample is generally tested with an external standard, an additive recovery, or a quality control sample. Therefore, as long as the signal-to-noise ratio obtained during the final inspection meets the detection limit, this step can be considered. Ignored for system errors.

2, activity phase

If the active phase consists of HPLC-grade solvents, the active phase does not need to be filtered. This is because HPLC-grade organic solvents, such as acetonitrile, methanol, etc., have been filtered through a 0.2 μm microfiltration membrane during the manufacturing process. Similarly, whether you are buying HPLC-grade water or using ultra-pure water purification systems in the laboratory, the final step is through 0.2 μm microfiltration membranes. However, if any solid solution such as phosphate is added to any of the buffers, active phase filtration will be a necessary step. Although the buffer salt may be soluble and highly pure, it may contain particulate matter. For example, when capping the plastic inner cap of the reagent bottle, the cap of the plastic bottle is pressed against the edge of the bottle mouth to generate plastic particles. In this case, a solid substance added may be completely dissolved, but a small amount of impurity particles exist in the active phase as a residue.

The filtration of the mobile phase through a 0.45 μm microfiltration membrane is an effective method for removing all particles from the mobile phase. 0.2μm microfiltration membranes can also be used, but they are not more effective than 0.45μm membrane filters for this application, and its filtration rate will be slower, especially when the quality of reagents and water used in the laboratory is not Good time. It is recommended that laboratories, when preparing standard operating procedures (SOPs) for the preparation of their activities, be able to interpret the requirements of similar laboratories in the world, namely:

The active phase preparation does not require filtration when using HPLC-grade liquids, whereas all active phase compositions must be filtered before use.

It is also important to use a submerged filter at the end of the infusion tube connecting the reservoir bottle and the pump (common materials are two types of molten glass core filter plates and microporous metals). The size of this filter is ≥10μm microporous material, so it can not replace the active phase filtration step, but it can remove the dust in the system and ensure the reliability of the use of liquid bottle, infusion tube.

3, the wear of the instrument system components

Finally, another major source of particulate matter in the HPLC system is the wear of the infusion pump seal and the injection valve rotating shaft. There are two different suggestions for changing the wear of the infusion pump seal.

The first suggestion is that the infusion pump seals in general laboratories usually have a service life of six months to one year. It is therefore recommended that these seals be replaced in half a year or one year. The laboratory should develop a regular preventive maintenance plan based on the above viewpoint. This point of view assumes that compared with the cost of replacing the column with the infusion pump gasket particles clogging the column, the cost of replacing the gasket is lower. Some infusion pumps have glass sand cores or screens that can filter out particles that have been abraded from the pump gasket in the flow path and prevent these particles from flowing with the active phase to the stigma. If there is such a device, consult the infusion pump operating manual for recommended intervals for cleaning or replacing the filter.

Another suggestion is that the original seal has the best sealing effect and easily causes leakage of the active phase after replacement. Therefore, do not easily replace the gasket as long as it does not leak.

Both arguments have their own rationale. How to manipulate them? It is recommended that you communicate with the instrument company's engineers. The instruments of each company are still somewhat different.

Sealing of the rotary shaft of the autosampler wears with use time, but in my experience, it can be used for several years even with high-load rotating shaft seals. If your autosampler system has the function of counting the number of rotations of the injection valve, you can set an alarm to warn you when the preset valve rotation has been reached. There was a claim that the injector could be rotated up to 20,000 times. This was just 10,000 injections; but this does not seem to be the typical sample analysis service life that the labs are involved with. Their actual service life will be longer. Rotary shaft seals will be exudate after wear. The most obvious feature is that after the same sample is injected multiple times, the peak area values ​​are quite different (RSD> 5%). Of course, the gasket wear of the infusion pump's seal and rotating shaft will add more abrasive to the active phase and accelerate damage to these components. In addition, if you are running buffered salts, such as phosphate-buffered salts, in your daily activities, the gasket will wear faster.

Regardless of what the particle originated from, it should be removed during the experiment. It is recommended to use a 0.45 or 0.5 μm on-line porous filter in the HPLC system between the autosampler and the column, even if the guard column is used. This in-line filter will act as a bezel instead of a stigma filter plate, and if a glass sand core filter plate is used, it is cheap and easy to replace (for a few minutes). If on-line filtration is used, the HPLC system will record the value of the pressure recorded before the beginning of each batch of samples. When the pressure rises by a certain value, for example, 25% or 500 psi, the glass sand core filter plate should be replaced. After several minutes of flushing, the system will recover. The pressure value.