How to Solve the Problem of Poor Repeatability of HPLC Column?
There are main reasons for the poor repeatability of the HPLC columns:
(1) The injection valve leaks;
(2) The sampling needle is not in place.
(3) Insufficient liquid volume.
(4) Column Degradation:
Causes: Over time, HPLC columns can degrade due to exposure to harsh solvents, high temperatures, or chemical interactions with samples. Contaminants or particulates in the sample can also contribute to column degradation.
Effects: Column degradation leads to changes in column efficiency, selectivity, and retention times. This can result in inconsistent and unpredictable chromatographic results.
(5) Improper Sample Preparation:
Causes: Inadequate sample preparation, such as incomplete dissolution, incomplete filtration, or improper concentration, can introduce variability in the injected sample. This can lead to erratic peak shapes and retention times.
Effects: Poorly prepared samples can lead to incomplete separation, distorted peaks, and unreliable results. It can also contribute to column fouling and contamination.
(6) Inconsistent Mobile Phase Preparation:
Causes: Variability in the preparation of the mobile phase, including inaccurate composition or pH adjustments, can significantly impact chromatographic results. Additionally, improper handling or storage of mobile phase components can lead to inconsistencies.
Effects: Changes in mobile phase composition alter the interactions between the stationary phase and the analytes, leading to shifts in retention times and peak shapes. This results in poor repeatability.
Addressing these issues involves careful attention to column maintenance, sample preparation, and mobile phase handling. Regular column conditioning, proper sample dissolution and filtration, and meticulous mobile phase preparation are crucial steps in maintaining repeatability in HPLC analyses. Additionally, routine monitoring, calibration, and validation procedures should be implemented to ensure consistent performance of the HPLC system.
To replace the injection valve gasket for this case; for the second case, ensure that the needle is inserted to the end, and the sample solution must be quickly and smoothly switched from the LOAD state to the INJECT state to ensure the injection volume is accurate. In daily work, the maintenance of the liquid chromatograph is very important. If you are careful not to let the air enter the infusion system and the high-pressure pump, the solution in the reservoir should be cleaned and replaced with a solution if it has not been used for a long time. After the chromatograph is finished, the buffer should be rinsed with pure water to prevent precipitation or deposition of inorganic salts. The pretreatment of the sample is also important. Any sample should be removed as much as possible, and completely dissolved, and the contamination of the HPLC columns should be minimized. Extend the service life of the column while avoiding the injection of the concentrated sample solution to prevent the residual liquid from clogging the injection valve. The column is marked and the column for different analysis purposes should not be mixed.