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:
Retention time drift in High-Performance Liquid Chromatography (HPLC) can be caused by several factors. Here are some common issues and troubleshooting steps:
Mobile Phase Composition:
Problem: Changes in mobile phase composition or pH can alter retention times.
Solution: Ensure the mobile phase is properly prepared and mixed. Check the pH if applicable.
Problem: Contaminants in the sample or column can lead to retention time shifts.
Solution: Flush the column with an appropriate solvent to remove contaminants. If necessary, consider replacing the column.
Problem: Over time, columns can degrade, leading to changes in retention times.
Solution: Regularly inspect and maintain columns. Replace the column if it shows signs of degradation.
Problem: Variations in column temperature can affect retention times.
Solution: Ensure that the column temperature is stable and within the recommended range.
Problem: Inconsistent sample preparation or matrix effects can cause retention time drift.
Solution: Standardize sample preparation methods and consider using an internal standard to account for matrix effects.
Flow Rate Variations:
Problem: Fluctuations in flow rate can impact retention times.
Solution: Verify that the HPLC system is properly calibrated and that the flow rate is stable.
Gradient Elution Issues:
Problem: Incorrect gradient program or mixing issues can lead to retention time shifts.
Solution: Verify the gradient program and ensure that the mobile phases are properly mixed.
Problem: Problems with the injector, such as sample overload or injector contamination, can cause retention time drift.
Solution: Clean or replace the injector components as needed.
Problem: Detector issues, such as drift or noise, can affect retention time accuracy.
Solution: Calibrate and maintain the detector regularly. Check for any anomalies in the detector output.
Problem: Variations in system pressure or stability can lead to retention time drift.
Solution: Regularly perform system suitability tests and calibrations to ensure stability.
Problem: Inadequate column equilibration can lead to unstable retention times.
Solution: Allow the column to equilibrate for a sufficient period before sample injection.
Problem: Using an inappropriate column for the application can lead to retention time drift.
Solution: Ensure that the column is suitable for the analytes being separated.
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.
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.
Always document any changes made during troubleshooting, and consider performing system suitability tests regularly to monitor performance. If issues persist, consult with the manufacturer or a chromatography expert for further guidance.