Experimental Technique | For volatile or highly viscous reagents, pay attention to the preparation of the mobile phase.

The mobile phase is a crucial aspect of high-performance liquid chromatography (HPLC), and the compliance and accuracy of its handling directly impact the precision and effectiveness of experimental results. In routine analysis, it’s common to encounter situations where the mobile phase contains volatile reagents (such as trifluoroacetic acid (TFA), triethylamine, concentrated ammonia, etc.), as well as components with higher viscosity (such as phosphoric acid). When preparing the mobile phase with these components, special attention is required in their addition methods to prevent potential effects on experimental results due to reagent evaporation or incomplete dispensing.

Triethylamine is a commonly used component in liquid-phase mobile phases for its role in pH adjustment, masking silanol groups on the stationary phase to modify peak shapes, and improving peak tailing, among other functions. However, it’s also a volatile reagent. When added conventionally above the liquid surface, inaccuracies in mobile phase preparation can occur due to the evaporation of triethylamine.

Therefore, when adding volatile reagents like triethylamine, it is recommended to use a bottom-dispensing pipette tip and dispense the reagent below the liquid surface to avoid inaccuracies in mobile phase composition caused by the evaporation of triethylamine. This practice helps ensure accurate and consistent mobile phase preparation.

Example:

For a certain project, the mobile phase consists of a 15mmol/L potassium dihydrogen phosphate solution (containing 0.06% triethylamine and 0.14% phosphoric acid).

Mobile Phase Preparation Method 1: Conventional approach, where triethylamine is dispensed above the liquid surface during mobile phase preparation.

Mobile Phase Preparation Method 2: Using a bottom-dispensing pipette tip, add triethylamine below the liquid surface.

By comparing the two diagrams above, it is evident that different mobile phase preparation methods can result in significant changes in peak retention times.

Conclusion

When preparing the mobile phase, it’s essential to choose appropriate preparation methods based on the characteristics of different reagents, as a one-size-fits-all approach is not suitable.

1. For volatile reagents such as triethylamine, diethylamine, trifluoroacetic acid, heptafluorobutyric acid, etc., when adding them, it’s advisable to insert the pipette tip below the liquid surface during preparation to prevent evaporation-induced concentration differences.
2. For viscous reagents like phosphoric acid, it’s recommended to dispense slowly during pipetting, and after pipetting, wipe the area around the pipette tip with a tissue to prevent reagent accumulation on the tip, which could affect the concentration. During addition, attention should be paid to a controlled liquid dispensing speed to avoid instances where part of the reagent adheres to the pipette wall and doesn’t flow down due to excessive speed, leading to concentration discrepancies in the mobile phase.