Troubleshooting HPLC Column Retention Time Drift – Part 2

(1) 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 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.

(2) 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.

(3) Hydrophobic collapse

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 amount of organic components, and then equilibration with a mobile phase of high water content. This phenomenon can also occur with 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.