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:
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 the column, causing drift in retention time. It should be noted that water is a mobile phase component that is easily contaminated.
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.
HPLC column contamination
Another common cause of retention time drift is HPLC column contamination. The HPLC column is a very effective adsorptive filter that filters and adsorbs any material carried by the mobile phase. Sources of contamination can be the mobile phase itself, mobile phase vessels, connecting tubes, pumps, injectors and instrument gaskets, and samples. The source of contamination can usually be determined experimentally.
The presence of strong components on the HPLC column in the sample may be a potential source of retention time drift. These roots are usually the sample matrix. Such as excipients in the drug, protein, and lipid compounds in biochemical samples (such as serum), the starch in food samples, humic acid in environmental water samples, etc. Usually, the strongly retained component in the sample has a higher molecular weight, in which case the retention time drifts or there is an increase in back pressure. The effect of the sample matrix can be removed by using a sample preparation method such as solid phase extraction (SPE).
The easiest way to avoid contamination with HPLC columns is to prevent them from happening. In contrast, finding the problem and designing an effective cleaning step to remove contaminants is much more difficult. Strong solvents are used under given chromatographic conditions, but not all contaminants can be dissolved in the mobile phase. For example, THF removes many of the contaminants in the reversed-phase column, but the protein does not dissolve in THF. DMSO is often used to remove proteins from reversed phase columns. Using a guard column is a very effective method. Recoil columns are only a last resort.
Mobile phase composition
Slow changes in the composition of the mobile phase are also common causes of retention time drift. For example, the volatile components in the mobile phase are volatilized and the flow in circulation is equal.
When a reverse-phase packed column with a good pore size and end-end sealing uses nearly 100% water as the mobile phase, a sudden loss of separation and a significant decrease or no retention of the analyte retention sometimes occur, which is a hydrophobic collapse. This phenomenon is caused by the fact that the mobile phase does not wet the surface of the stationary phase. The salvage method achieves infiltration of the stationary phase with a mobile phase containing a large number of organic components, and then equilibration with a mobile phase of high water content. This phenomenon can also occur with the long-term storage of HPLC columns. Collapsing can also be avoided by using a reversed-phase column with a non-polar group or a non-end-sealed column.