What? Method Reproducibility Again! (Part 2)

Poor durability of detection methods

Solvent effect

When the ratio of the solvent and the mobile phase starting organic phase is different, the detection effect will be different on different instruments.

The picture shows the detection effect of the same project on two different instruments. The solvent for this project was 50% acetonitrile, and the starting ratio of the mobile phase was 25% acetonitrile. Figure 1 shows the effect on an instrument with a larger inner diameter of the pre-column pipeline, and Figure 2 shows the effect on an instrument with a smaller inner diameter. The larger inner diameter of the tubing allows the sample to be thoroughly mixed before entering the column, eliminating solvent effects and presenting better peak shapes. This kind of project with solvent effect can be solved by using the instrument with thicker pipeline or reducing the injection volume. Of course, the safer method is to replace the sample solvent with the polarity closer to the initial mobile phase. solvent.

Extra-column effect

If the injection volume is too large, the volume between the injection valve-chromatographic column-detector, the detection cell, and the joint will be large, which will cause extra-column effects and lead to abnormal peak shapes. Therefore, for such samples, the requirements for the instrument should be more detailed in the method.

Mobile phase composition changes

Improper buffer selection, such as buffer salt concentrations that are too low or out of the effective buffer pH range, can result in poor reproducibility. For some samples that are particularly sensitive to pH, slight changes in the pH of the mobile phase can lead to unreproducible results. For such samples, special attention should be paid to the buffer range and concentration of the buffer when selecting the buffer to ensure that the mobile phase system can provide a stable pH value for sample detection.

Some samples are particularly sensitive to changes in the organic phase. For these samples, it is recommended to choose online mixing of the mobile phase.

Conditions do not control column temperature

Many samples will have different selectivities due to changes in column temperature. Therefore, if the column temperature is not controlled, it can lead to reproducibility problems of the sample due to the change of the column temperature. It is recommended to verify the durability of the column temperature at a temperature of ±10 °C during method development. If the column temperature is sensitive to changes in column temperature, the detection column temperature should be included in the method.

Other questions

In addition, problems such as slow column equilibration, column overload, and instrument failure can also make the method unreproducible.