I believe that most laboratory workers have more or less encountered an experimental phenomenon that makes everyone more headaches in daily testing: when we continuously inject samples, the retention time of the sample cannot be reproduced. For retention time skew and retention time fluctuation. Generally, when looking for the cause, it is very helpful to distinguish the two situations and investigate the cause of the problem.

Some of the most common causes of sample retention time drift and retention time usually fluctuations

Column equilibration

If we observe retention time skew, the first consideration should be whether the column has been fully equilibrated with the mobile phase. Usually equilibration requires 10-20 column volumes of mobile phase, but if a small amount of additives (such as ion-pair reagents) are added to the mobile phase, it will take quite a long time to equilibrate the column.

Stationary Phase Stability

The stability of the stationary phase is also one of the reasons for the drift of the retention time. The stability of the stationary phase is limited. Even if it is used within the recommended pH range, the stationary phase will slowly hydrolyze. For example, a silica matrix has the best hydrolytic stability at pH 4. The rate of hydrolysis is related to mobile phase type and ligand. Bifunctional ligands and trifunctional ligands are more stable than the bonded phase of monofunctional ligands; long chain bonded phases are more stable than short chain bonded phases; alkyl bonded phases are much more stable than cyano bonded phases. Other silica-based bonded phases can also undergo hydrolysis in high-volume aqueous environments, such as amino-bonded phases.

Mobile phase composition

Slow changes in mobile phase composition are also a common cause of retention time drift. For example, the volatilization of volatile components in the mobile phase and the recycling flow are equal. Mobile phase contamination may also be a cause. Small amounts of contaminants dissolved in the mobile phase may slowly accumulate on the column, causing retention time drifts. It is recommended to replace with new mobile phase in time.

Hydrophobic collapse

When the reversed-phase packing column uses nearly 100% water as the mobile phase, sometimes the sudden loss of separation and the retention of the analyte are significantly reduced or not retained at all, which is hydrophobic collapse. This phenomenon is caused by the mobile phase not wetting the surface of the stationary phase. Hydrophobic collapse of the column is also a common cause of retention time skew. Conventional C18 columns can be used normally in about 95% of the aqueous environment without hydrophobic collapse. If it is used in pure water, it is recommended to use a modified special water-resistant column for the experiment (such as Welch’s Ultisil ® AQ-C18).

Column contamination

Another common cause of retention time drift is column contamination. If the method has been established, there is no problem at the beginning, and this problem occurs after a period of use, which may be caused by column contamination. In this case, the column needs to be intensively flushed, that is, the column is flushed with a stronger solvent than the mobile phase of the method. It is also possible to add a guard column in advance when the test sample matrix is dirty.

Other factors

If there are air bubbles in the pump or pipeline, the retention time will drift back and forth, and the air bubbles can be driven out by exhaust, etc. The jewel ball in the pump check valve is worn, causing the mobile phase to flow backwards, the pressure is unstable, and the retention time will drift back and forth. The solution is to replace the parts. The control of the proportional valve of the pump fails, the proportion of the flow phase in different flow paths changes constantly, and the retention time will drift back and forth. The solution is to replace the parts. The pump piston rod seal is aging and the mobile phase is leaking. The solution is to change the gasket. The mixing ability of the online mixer becomes worse, the mixing ability is not connected to the gradient test, and compared with the manual configuration flow.

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