We flush our HPLC column with methanol followed by filtered deionised water and finally with mobile phase(ammonium acetate + glacial acetic acid + DI water) before we start running samples. Why is it necessary to flush with filtered DI water before flushing with mobile phase? Why can't we flush with methanol and go straight to flushing column with mobile phase?
(we run our samples using gradient elution- methanol for pump A and mobile phase for pump B)
Flushing an HPLC column with methanol before running samples is a common practice to remove any residual contaminants, impurities, or analytes from the previous analysis, effectively cleaning the column. Methanol has a strong solvent power and can effectively dissolve various compounds, making it an ideal choice for this purpose.
Filtering deionized water is typically done after methanol flushing to further rinse the column. The purpose of using filtered DI water is to remove any remaining methanol from the column. While methanol is a useful solvent, it can cause unwanted interactions with the mobile phase, affecting the separation and detection of analytes. By rinsing the column with filtered DI water, it helps to eliminate any traces of methanol, ensuring that it does not interfere with the subsequent mobile phase.
While it may seem feasible to flush the column with methanol and then go straight to flushing with the mobile phase, there are a few reasons why it is not usually done. Firstly, directly transitioning from methanol to the mobile phase can lead to a mixing of the solvents. This can cause changes in the composition of the mobile phase, potentially altering the elution properties and compromising the separation of analytes.
Secondly, methanol and the mobile phase might have different flow properties, such as viscosity or solvent strength. Skipping the DI water rinse could result in residual methanol affecting the flow characteristics of the mobile phase in an unpredictable manner. This can lead to suboptimal separations and negatively impact the reproducibility and accuracy of the analysis.
Finally, methanol is a strong solvent, and some samples may have compounds that are very soluble in methanol but less soluble in the mobile phase. If the column is not adequately rinsed with DI water, these compounds may not be fully removed, leading to possible carryover into subsequent analyses.
In summary, flushing an HPLC column with filtered DI water after methanol flushing is essential to remove any residual methanol and prevent potential adverse effects on separation and accuracy. It ensures the column is thoroughly cleaned and ready for proper analysis using the mobile phase.