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)
is pbcl2 homogeneous or heterogenous?
The reason we flush our HPLC column with filtered deionized (DI) water before flushing it with the mobile phase is to ensure proper equilibration and conditioning of the column before running the samples. Here's why this step is necessary:
1. Removal of Methanol: Methanol is typically used for cleaning and conditioning the column between runs. However, methanol is not a suitable solvent for sample analysis as it can interfere with the detection method or affect the separation. Therefore, we need to flush it out before introducing the sample.
2. Elimination of Contaminants: Methanol and other solvents used in HPLC can leave traces of impurities in the column. Flushing with DI water helps to remove these contaminants, ensuring that the column is clean and ready for sample analysis.
3. Preparation for Mobile Phase: Flushing the column with DI water after methanol helps to prepare it for the mobile phase. DI water helps to stabilize the pH and ionic strength of the column, bringing it closer to the conditions that will be used during the sample analysis.
4. Column Equilibration: Equilibration is an important step in HPLC, especially when running gradients. By flushing the column with DI water, we allow it to reach a stable baseline and ensure consistent results before introducing the mobile phase.
5. Prevention of Sample Carryover: Flushing the column with DI water after methanol helps to minimize the chances of sample carryover from previous runs. Water can act as a barrier between different samples or contaminants, reducing the risk of cross-contamination.
In summary, flushing with filtered DI water after methanol ensures the removal of any remaining impurities or contaminants, prepares the column for the mobile phase, and helps achieve a stable baseline. It is a critical step in the HPLC method to ensure accurate and reproducible results.