Troubleshooting HPLC Column Retention Time Drift
There are two different cases of retention time: retention time drift and retention time fluctuations. The former means that the retention time changes only in one direction, while the latter refers to the fluctuation of the retention time without a fixed law. It is often helpful to distinguish between the two situations to find the cause of the problem. The most common reasons for retention time drift are as follows:
(1) HPLC column balance
If we observe retention time drift, we should first consider whether the HPLC column is completely equilibrated with the mobile phase. Usually, a balance of 10-20 column volumes of the mobile phase is required, but if a small amount of additive (such as an ion pair reagent) is added to the mobile phase, it takes a considerable amount of time to equilibrate the column.
Mobile phase contamination may also be one of the reasons. A small number of contaminants dissolved in the mobile phase may slowly enrich on the column, causing drift in retention time. It should be noted that water is a mobile phase component that is easily contaminated.
(2) Stationary phase stability
The stability of the stationary phase is limited and the stationary phase will slowly hydrolyze even when used within the recommended pH range. For example, the silica gel matrix has the best hydrolytic stability at pH 4. The rate of hydrolysis is related to the type of mobile phase and the ligand. The bifunctional ligand and the trifunctional ligand are more stable than the bonded phase of the monofunctional ligand; the long chain linkage is more stable than the short chain bonding phase; the alkyl linkage is much more stable than the cyano bonded phase.
Frequent cleaning of the HPLC column also accelerates the hydrolysis of the HPLC column stationary phase. Other silica matrix bonded phases may also undergo hydrolysis in aqueous solutions, such as amino bonding.