Troubleshooting of HPLC retention time drift

The retention time does not recur in two different situations: retention time drift and retention time fluctuation. 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 these two situations to find the cause of the problem. For example, retention time drift is often caused by column aging; and column aging is unlikely to cause retention and retention time does not reappear in two different situations: retention time drift and retention time fluctuation. 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 these two situations to find the cause of the problem. For example, the drift of retention time is often caused by column aging; and column aging is unlikely to cause irregular fluctuations in retention time. In fact, most of the reasons for retention time drift are column aging due to different mechanisms, such as stationary phase loss (for example, through hydrolysis), column contamination (due to sample or mobile phase), etc. The most common reasons for retention time drift are as follows: 　　一 Column Equilibrium　　 If we observe retention time drift, we should first consider whether the column has been completely balanced with the mobile phase. Normally, 10-20 column volumes of the mobile phase are required for equilibrium, but if a small amount of additives (such as ion pair reagents) are added to the mobile phase, it will take a long time to equilibrate the column.　　 Mobile phase contamination may also be one of the reasons. A small amount of contaminants dissolved in the mobile phase may slowly accumulate on the chromatographic column, causing retention time drift. It should be noted that water is a mobile phase component that is easily contaminated.　　二Stability of the stationary phase 　　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, 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 ligand. Bifunctional ligands and trifunctional ligands are more stable than monofunctional ligands in the bonded phase; long-chain bonding is more stable than short-chain bonding; alkyl bonding is much more stable than cyano-bonded phases.　　 Frequent cleaning of the chromatographic column will also accelerate the hydrolysis of the stationary phase of the chromatographic column. Other silica-based bonded phases can also be hydrolyzed in an aqueous environment, such as amino bonding.　　三 Column contamination! Another common cause of retention time drift is column contamination. The HPLC column is a very effective adsorptive filter, which can filter and adsorb any substance carried by the mobile phase. Contamination sources can be: the mobile phase itself, the mobile phase container, connecting pipes, pumps, injectors and instrument gaskets, and samples. The source of pollution can usually be determined through experiments.　　 If there are components that are strongly retained on the chromatographic column in the sample, it may be the potential source of the retention time drift. These root causes are usually the sample matrix. Such as: excipients in pharmaceutical preparations, proteins and lipid compounds in biochemical samples (such as serum), starch in food samples, humic acid in environmental water samples, etc. Generally, the strongly retained components in the sample have a higher molecular weight. In this case, the back pressure will increase at the same time or after the retention time drift. The influence of sample matrix can be removed by using sample preparation methods such as solid phase extraction (SPE).　　 The easiest way to avoid column contamination is to take precautions. In contrast, finding the source of the problem and designing effective cleaning steps to remove contaminants is much more difficult. Usually strong solvents under given chromatographic conditions are used, but not all contaminants can be dissolved in the mobile phase. For example, THF can remove many contaminants in reversed-phase chromatography columns, but proteins cannot be dissolved in THF. DMSO is often used to remove proteins from reversed-phase chromatography columns.　　Using a guard column is a very effective method. Backflushing the chromatographic column is only a last resort.　　4 Mobile phase composition 　　 The slow change of mobile phase composition is also a common cause of retention time drift. For example, the volatilization and circulation of the volatile components in the mobile phase are equal. 5. Hydrophobic collapse. When a reversed-phase packing chromatographic column with small pore size and good end-group sealing uses close to 100% water as the mobile phase, the separation may be suddenly lost and the retention of the analyte may be significantly reduced or not retained at all. This is Hydrophobic collapse. This phenomenon is caused by the mobile phase not infiltrating the surface of the stationary phase. The rescue method is to infiltrate the stationary phase with a mobile phase containing a large amount of organic components, and then balance it with a mobile phase with high water content. This phenomenon can also occur in long-term storage of the chromatographic column. The use of reversed-phase columns with embedded polar groups (such as Waters SymmetryShield RP columns) or non-end-capped columns (such as Waters Resolve columns) can also avoid collapse.